InteractiveFly: GeneBrief

gawky: Biological Overview | References

BIOLOGICAL OVERVIEW

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the RNA-binding protein GW182, which interacts with the AGOs and is required for miRNA function. Depletion of Drosophila GW182 (Gawky), leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1 and NOT1, components of a deadenylase complex, or the DCP1:DCP2 decapping protein complex. The N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. These findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets (Behm-Ansmant, 2006).

To accomplish their regulatory function miRNAs associate with the Argonaute proteins to form RNA-induced silencing complexes (RISCs), which elicit decay or translational repression of complementary mRNA targets. In plants, miRNAs are often fully complementary to their targets, and elicit mRNA decay. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that remain elusive. Recent studies have shown that miRNAs silence gene expression by inhibiting translation initiation at an early stage involving the cap structure; mRNAs translated via cap-independent mechanisms escape miRNA-mediated silencing. Other studies have suggested that translation inhibition occurs after initiation, based on the observation that miRNAs and some targets remain associated with polysomes. In addition, animal miRNAs can also induce significant degradation of mRNA targets despite imperfect mRNA-miRNA base-pairing (Behm-Ansmant, 2006 and references therein).

The existence of a link between the miRNA pathway and mRNA decay is supported by the observation that mammalian Argonaute proteins (AGO1-AGO4), miRNAs, and miRNA targets colocalize to cytoplasmic foci known as P-bodies. These mRNA processing bodies are discrete cytoplasmic domains where proteins required for bulk mRNA degradation in the 5'-to-3' direction accumulate (e.g., the decapping DCP1:DCP2 complex and the 5'-to-3' exonuclease XRN1). Additional components of P-bodies in yeast and/or human cells include the CCR4:NOT deadenylase complex (see Drosophila ), auxiliary decapping factors (e.g., the LSm1-7 complex and Pat1p/Mtr1p), the cap-binding protein eIF4E, and the RNA helicase Dhh1/Me31B involved in translational repression. In metazoa, P-bodies are also marked by the presence of GW182, a protein with glycine-tryptophan repeats (GW repeats) required for P-body integrity (Behm-Ansmant, 2006 and references therein).

The presence of Argonaute proteins, miRNAs, and miRNA targets in P-bodies has led to a model in which translationally silenced mRNAs are sequestered to these bodies, where they may undergo decay. At present, it is unclear whether the localization in P-bodies is the cause or consequence of the translational repression, though several lines of evidence point to a direct role for P-body components in miRNA-mediated gene silencing. (1) DCP1, GW182, and its paralog TNRC6B associate with AGO1 and AGO2 in human cells; (2) depletion of GW182 in human cells impairs both miRNA function and mRNA decay triggered by complementary short interfering RNAs (siRNAs). Similarly, miRNA function is impaired in Drosophila Schneider cells (S2 cells) depleted of GW182 or the decapping DCP1:DCP2 complex (Rehwinkel, 2005). (3) The Caenorhabditis elegans protein AIN-1, which is related to GW182, is required for gene regulation by at least a subset of miRNAs (Behm-Ansmant, 2006 and references therein).

In Drosophila, siRNA-guided endonucleolytic cleavage of mRNAs (RNA interference [RNAi]) is mediated by AGO2, while gene silencing by miRNAs is mediated by AGO1. That siRNAs and miRNAs enter separate pathways in Drosophila is further supported by the observation that depletion of GW182 inhibits miRNA-mediated, but not siRNA-mediated gene silencing (Rehwinkel, 2005). The precise role of GW182 in the miRNA pathway is unknown. GW182 could have an indirect role by affecting P-body integrity. Alternatively, it could be more directly involved, localizing miRNA targets to P-bodies or facilitating the mRNP remodeling steps required for the silencing and/or decay of these targets (Behm-Ansmant, 2006 and references therein).

This study investigates the role of Drosophila GW182 in the miRNA pathway. Depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of AGO1, indicating that GW182 is a genuine component of the miRNA pathway. In cells in which miRNA-mediated gene silencing is suppressed by depletion of AGO1, GW182 can still silence the expression of bound mRNAs, suggesting that GW182 acts downstream of AGO1. It is further shown that GW182 triggers silencing of bound transcripts by inhibiting protein expression and promoting mRNA decay via a deadenylation and decapping mechanism. Finally, evidence is provided that mRNA degradation by miRNAs requires GW182, the CCR4:NOT deadenylase, and the DCP1:DCP2 decapping complexes. Together with the observation that GW182 interacts with AGO1, these results indicate that binding of GW182 to miRNA targets induces silencing and can trigger mRNA degradation, providing an explanation for the observed changes in mRNA levels, at least for a subset of animal miRNA targets (Behm-Ansmant, 2006).

These results indicate that GW182 is a genuine component of RNA silencing pathways, associating with the Argonaute proteins and with components of the mRNA decay machinery and, providing a molecular link between RNA silencing and mRNA degradation. Depletion of GW182 or AGO1 from Drosophila cells leads to correlated changes in mRNA expression profiles, indicating that these proteins act in the same pathway. Transcripts commonly up-regulated by AGO1 and GW182 are enriched in predicted and validated miRNA targets. These results, together with the observation that GW182 associates with AGO1, identify GW182 as a component of the miRNA pathway (Behm-Ansmant, 2006).

GW182 belongs to a protein family with GW repeats, a central UBA domain, and a C-terminal RRM. Multiple sequence alignment of all proteins possessing these domains revealed that there are three paralogs (TNRC6A/GW182, TNRC6B, and TNRC6C) in vertebrates, a single ortholog in insects, and no orthologs in worms or fungi. At present, it is unclear whether the vertebrate paralogs have redundant functions, but both GW182 and TNRC6B have been shown to associate with human AGO1 and AGO2 (Behm-Ansmant, 2006).

In Drosophila, GW182 interacts with AGO1 in vivo and in vitro. No stable interaction with AGO2 was detected under the same conditions, suggesting that AGO2 may act independently of GW182. This is consistent with the observation that depletion of GW182 does not affect siRNA-guided mRNA cleavage or RNAi, which is mediated exclusively by AGO2 in Drosophila. Nevertheless, since AGO2 also regulates the expression levels of a subset of miRNA targets (Rehwinkel, 2006), the lack of interaction with GW182 raises the question of whether this regulation occurs by a similar or different mechanism from that mediated by AGO1. Further studies are needed to elucidate the mechanism by which Drosophila AGO2 regulates the expression of a subset of miRNA targets (Behm-Ansmant, 2006).

The N-terminal GW repeat region of GW182 encompasses two highly conserved motifs (I and II) and is expanded in vertebrates. This region is shorter in insects and bears similarity to the GW-like regions in the C. elegans protein AIN-1, involved in the miRNA pathway. However, AIN-1 does not contain UBA, Q-rich, or RRM domains. This lack of common domain architecture suggests that AIN-1 represents a functional analog. Nevertheless, the observation that C. elegans AIN-1 also localizes to P-bodies and interacts with AGO1 (i.e., worm ALG-1), and the finding that the N-terminal GW repeats of Drosophila GW182 interact with the PIWI domain of AGO1, suggest a conserved role for these repeats in mediating the interaction with Argonaute proteins. It would be of interest to determine the molecular basis of the specific interaction between the N-terminal GW repeats of GW182 and the PIWI domain of AGOs, and whether this interaction affects the catalytical activity of the domain (Behm-Ansmant, 2006).

Apart from the interaction with AGO1, the N-terminal repeats and the UBA and Q-rich domains contribute to the localization of GW182 in P-bodies, which is in turn required for P-body integrity. This suggests that GW182 may act as a molecular scaffold bringing together AGO1-containing RISCs and mRNA decay enzymes, possibly nucleating the assembly of P-bodies. Understanding the precise role of the various GW182 domains in the interaction with mRNA decay enzymes and AGO1 as well as in P-body integrity awaits further biochemical characterization (Behm-Ansmant, 2006).

Tethering GW182 to a reporter transcript silences its expression, bypassing the requirement for AGO1. Silencing by GW182 occurs by two distinct mechanisms: repression of protein expression, and mRNA degradation. It remains to be elucidated how GW182 represses translation. mRNA degradation by GW182 is inhibited in cells depleted of CAF1, NOT1, or the DCP1:DCP2 complex, indicating that GW182 promotes mRNA deadenylation and decapping. Thus, binding of GW182 appears to be a point of no return, which marks transcripts as targets for degradation (Behm-Ansmant, 2006).

More studies are needed to determine whether decapping triggered by GW182 requires prior deadenylation or whether these two events occur independently. The observation that mRNA levels are fully restored in cells depleted of DCP1:DCP2, suggests that deadenylation followed by 3'-to-5' exonucleolytic degradation is unlikely to represent a major pathway by which these mRNAs are degraded. Future studies should also reveal the identity of the nuclease(s) acting downstream of the decapping enzymes (Behm-Ansmant, 2006).

Previous studies indicate that miRNAs can reduce the levels of the targeted transcripts, and not just the expression of the translated protein. Consistently, transcripts up-regulated in cells depleted of AGO1 or GW182 are enriched in predicted and validated miRNA targets. In this paper further evidence is provided indicating that miRNAs silence gene expression by two mechanisms: one mechanism involving translational silencing, and one involving mRNA degradation. The contribution of these mechanisms to miRNA-mediated gene silencing appears to differ for each miRNA:target pair. Indeed, of the three reporters analyzed, Nerfin is silenced mainly at the translational level, silencing of the CG10011 reporter can be attributed to mRNA degradation, while Vha68-1 is regulated both at the translational and mRNA levels. Regardless of the extent of the contribution of these two mechanisms to silencing, both require AGO1 and GW182, because the levels of the mRNA reporter and luciferase activity are restored in cells depleted of any of these two proteins (Behm-Ansmant, 2006).

In contrast, although the levels of the mRNA reporter are restored in cells depleted of CAF1 or NOT1, translational repression is not fully relieved, indicating that deadenylation is required for mRNA decay, but not for translational silencing by miRNAs. In agreement with this, two reports published while this manuscript was in preparation have shown that miRNAs trigger accelerated deadenylation of their targets (Giraldez, 2006; Wu, 2006). This study extends these observations further by demonstrating: (1) deadenylation is mediated by the CCR4:NOT complex; (2) decapping is also required for miRNA target degradation, and (3) both deadenylation and decapping triggered by miRNAs requires GW182 (Behm-Ansmant, 2006).

Based on the results presented in this study and the observations that GW182 associates with AGO1 and is required for miRNA-mediated gene silencing, the following model is proposed: AGO1-containing RISCs binds to mRNA targets by means of base-pairing interactions with miRNAs; AGO1 may then recruit GW182, which marks the transcripts as targets for decay via a deadenylation and decapping mechanism (Behm-Ansmant, 2006).

A question that remains open is whether miRNA-mediated translational repression is the cause of mRNA degradation or whether these represent two independent mechanism by which miRNAs silence gene expression as proposed by Wu (2006). Indeed, changes in mRNA levels are not observed for all miRNA targets (Rehwinkel, 2006), suggesting that inhibition of translation is not always followed by mRNA decay. Conversely, depletion of CAF1 or NOT1 prevents mRNA decay but does not relieve translational silencing, suggesting that these two processes are independent (Behm-Ansmant, 2006).

An important finding is that miRNAs elicit degradation to different extents. One possible explanation is that the extent of degradation depends on the stability of the miRNA:mRNA duplexes. Also, the extent of degradation might depend on the particular set of proteins associated with a given target. For instance, some targets may assemble with a set of proteins that antagonize degradation. Finally, GW182 might interact only with a subset of AGO1-containing RISCs, as suggested for AIN-1. A major challenge will be to identify the specific features of miRNA targets and/or RISC complexes that lead to regulation of gene expression at the level of translation or at the level of mRNA stability (Behm-Ansmant, 2006).

MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. Previous work has shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. This study examined the assembly of translation initiation factors on the target mRNA under repression. Ago1-RISC was shown to induce dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block the assembly of the eIF4F complex during translation initiation (Fukaya, 2014).

MicroRNAs (miRNAs) silence their complementary target mRNAs via formation of the effector ribonucleoprotein complex called RNA-induced silencing complex (RISC). The core component of RISC is a member of the Argonaute (Ago) proteins. In Drosophila, miRNAs are sorted into two functionally distinct Ago proteins, Ago1 and Ago2, according to their structural features and the identity of the 5' end nucleotides. Compared to fly Ago2, fly Ago1 shares more common features with mammalian Ago1-4, making it a suitable model for investigating miRNA-mediated gene silencing in animals. Ago1-RISC mediates translational repression as well as shortening of the poly(A) tail followed by mRNA decay. While deadenylation per se disrupts the closed-loop configuration of mRNA and leads to inhibition of translation initiation, Ago1-RISC can repress translation independently of deadenylation. Such a deadenylation-independent 'pure' translational repression mechanism seems to be widely conserved among species (Fukaya, 2014).

Ago is not the only protein involved in the miRNA-mediated gene silencing pathway. In flies, a P-body protein GW182 specifically interacts with Ago1, but not with Ago2, through the N-terminal glycine/tryptophan (GW) repeats and provides a binding platform for PAN2-PAN3 and CCR4-NOT deadenylase complexes (Braun, 2011, Chekulaeva, 2011). This protein interaction network is conserved in animals including zebrafish, nematodes, and humans. Accordingly, GW182 is essential for shortening of the poly(A) tail by miRNAs. On the other hand, recent studies revealed that miRNA-mediated translational repression occurs in both GW182-dependent and -independent manners. Previous sedimentation analysis on sucrose density gradient suggested that both of the two translational repression mechanisms block recruitment of the ribosomal 43S preinitiation complex to the target mRNA independently of deadenylation (Fukaya, 2014).

In eukaryotes, recruitment of the 43S preinitiation complex is initiated by the formation of eukaryotic translation initiation factor 4F (eIF4F). eIF4F is a multiprotein complex composed of the cap-binding protein eIF4E, which recognizes the 7-methyl guanosine (m7G) structure of the capped mRNA; the scaffold protein eIF4G, which interacts with 40S ribosome-associated eIF3 and bridges the mRNA and the 43S preinitiation complex; and the DEAD-box RNA helicase eIF4A, which plays a pivotal role in translation initiation supposedly through unwinding the secondary structure of the 5' UTR for landing of the 43S complex. In addition, the poly(A)-binding protein PABP stimulates translation initiation through its direct interaction with eIF4G. miRNAs likely block one (or more) of these steps to repress translation initiation. It was recently proposed that, in mammals, preferential recruitment of eIF4AII-one of the two eIF4A paralogs-is required for miRNA-mediated translational repression (Meijer, 2013). This model postulates that eIF4AII acts to inhibit rather than activate translation, unlike its major counterpart eIF4AI. However, the role of eIF4AII in translation remains largely unexplored, as opposed to eIF4AI's well-established function to promote translation. Moreover, invertebrates have only one eIF4A, making this model incompatible in flies. Thus, it still remains unclear how miRNAs repress translation initiation. This is largely due to technical limitations in directly monitoring the assembly of the translation initiation complex specifically on the mRNA targeted by miRNAs (Fukaya, 2014).

By using site-specific UV crosslinking, this study examined the association of translation initiation factors on the target RNA under repression. Fly Ago1-RISC was shown to specifically induce dissociation of eIF4A from the target mRNA without affecting the 5' cap recognition by eIF4E in a manner independent of GW182 or PABP. On the other hand, direct tethering of GW182 to the target mRNA promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block assembly of the eIF4F complex during translation initiation, in addition to their established role in deadenylation and decay of their target mRNAs (Fukaya, 2014).

Thus fly Ago1-RISC induces dissociation of eIF4A without affecting the cap recognition by eIF4E. Although it was not possible to detect eIF4G via any of the crosslinking positions spanning from 2 nt to 13 nt downstream of the cap, it was previously shown that noncanonical translation driven by direct tethering of eIF4G to the 5' UTR was fully susceptible to translational repression by Ago1-RISC (Fukaya, 2012). Therefore, it was reasoned that Ago1-RISC directly targets eIF4A rather than eIF4E or eIF4G. In the accompanying paper, Fukao (2014) revealed that human Ago2-RISC specifically induces dissociation of eIF4A-both eIF4AI and eIF4AII-without affecting eIF4E or eIF4G in a cell-free system deriving from HEK293F cells. Thus, eIF4A is likely a target of miRNA action conserved among species. In agreement with this model, miRNA-mediated gene silencing is cancelled by the eIF4A inhibitors silvestrol (Fukao, 2014), hippuristanol, or pateamine A in human cells (Fukaya, 2014).

GW182 is a well-known interactor of miRNA-associated Ago proteins and is a prerequisite for miRNA-mediated deadenylation/decay of target mRNAs. GW182 directly binds to both NOT1 and CAF40/CNOT9, thereby recruiting the CCR4-NOT deadenylase complex to the target mRNA. It has been suggested that the CCR4-NOT complex not only shortens the poly(A) tail but also plays a role in miRNA-mediated translational repression, because direct tethering of the CCR4-NOT complex was capable of inducing translational repression independently of deadenylation. It was originally proposed that, in humans, the CCR4-NOT complex specifically binds to eIF4AII (but not to eIF4AI) to repress translation. However, this model was challenged by recent studies showing that, although the MIFG4 domain of human CNOT1 structurally resembles the middle domain of eIF4G, it does not bind eIF4AI or II but instead partners with the DEAD-box RNA helicase DDX6, which has been implicated in repression of translation initiation and/or translation elongation as well as activation of decapping. Given that miRNAs mediate gene silencing via multiple different pathways, recruitment of DDX6 by GW182 via the CCR4-NOT complex may well play a role in inhibiting protein synthesis from miRNA targets. Indeed, this study observed strong dissociation of both eIF4E and eIF4A by direct tethering of GW182. However, at the physiological stoichiometry between Ago1 and GW182 in S2 cell lysate, eIF4A was specifically dissociated without apparent effect on eIF4E by canonical miRNA targeting, which is in agreement with the result of the reporter assay in S2 cells depleted of each eIF4F component. It is envisioned that, although GW182 is clearly essential for miRNA-mediated deadenylation, the degree of contribution of GW182 to translational repression can vary in different cell types and conditions, depending on the concentrations of GW182 and Ago proteins, as well as their protein interaction networks that are subject to regulation by extracellular signaling. In this regard, direct tethering of GW182 may potentially overestimate its role in miRNA-mediated translational repression (Fukaya, 2014).

How could Ago1-RISC specifically dissociate eIF4A from the initiation complex? Previous studies hade shown that none of GW182, the CCR4-NOT complex, or PABP is required for translational repression by Ago1-RISC (Fukaya, 2012). The current data extend these findings to reveal that Ago1-RISC can induce dissociation of eIF4A independently of GW182 or PABP. It is tempting to speculate that an as-yet-unidentified factor associated with Ago1-RISC, or perhaps Ago1-RISC itself, blocks the interaction between eIF4G and eIF4A (e.g., similarly to Programmed Cell Death 4 [PDCD4] whose tandem MA-3 domains compete with the MA-3 domain of eIF4G to bind the N-terminal domain of eIF4A, thereby displacing eIF4A from the eIF4F initiation complex). Alternatively, Ago1-RISC might directly or indirectly inhibit the ATP-dependent RNA-binding activity of eIF4A, which is tightly regulated by its accessory proteins eIF4B and eIF4H (Abramson, 1988, Richter, 1999). Future studies are warranted to determine how miRNAs block the assembly of the eIF4F translation initiation complex (Fukaya, 2014).

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. This study characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which was named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, since human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development (Schneider, 2006).

The GW182 protein is a critical component of cytoplasmic RNP bodies that have been shown to function in mRNA degradation, storage, and, recently, microRNA (miRNA)- and siRNA-based gene silencing (Eystathioy, 2003; Yang, 2004; Ding, 2005; Jakymiw, 2005; Liu, 2005a; Rehwinkel, 2005). GW182 was named for the presence of multiple glycine (G)-tryptophan (W) amino acid pairs in the N-terminal region of a 182-kD protein with a predicted C-terminal RNA recognition motif (RRM). It localizes into cytoplasmic GW bodies (GWBs; Eystathioy, 2002; Maris, 2005) that also contain factors involved in 5'-3' mRNA decay, including the exonuclease XRN1, decapping enzymes DCP1 and DCP2, and the LSm1-7 decapping activator, pointing to a role for GWBs in regulating mRNA stability (Ingelfinger, 2002; Eystathioy, 2003; Cougot, 2004). These bodies may participate in additional roles in mRNA regulation, since they also contain the m7G cap-binding protein eIF4E and the eIF4E transporter but no other components of translation machinery. Importantly, intact GWBs are required for the functioning of the RNAi pathway in human cells potentially via direct interaction between GW182 (and the related TNRC6B protein) and Argonaute1 (Ago1) and 2 (Ago2; Jakymiw, 2005; Liu, 2005a,b; Meister, 2005; Schneider, 2006 and references therein).

GWBs are thought to be analogous to Saccharomyces cerevisiae cytoplasmic processing bodies (PBs). They are involved in mRNA decapping and 5'-3' exonucleolytic decay, and their integrity depends on the presence of nontranslating mRNAs. Both PBs and GWBs dissociate when polysomes are stabilized with drugs such as cycloheximide. However, despite similar compositions, there are functional differences between GWBs and PBs. GWBs increase in size and number in proliferating cells, whereas PBs increase in size and number during growth limitation and increased cell density. GWBs and PBs also differ in their responses to stress, as PBs increase in size and number in response to environmental stress. This is likely caused by decreased translation initiation because this response can be reproduced using a temperature-sensitive allele of Prt1p, a subunit of the eIF3 complex. In stressed mammalian cells, stalled preinitiation complex mRNAs are first targeted to stress granules (SGs), which may function as triage sites where mRNAs are sorted for future degradation, storage, or reinitiation of translation. Observation of interactions between SGs and GWBs in live cells suggest that transcripts may be exported from SGs to GWBs for degradation (Schneider, 2006 and references therein).

This study characterized the role of gawky (gw), the Drosophila melanogaster orthologue of the human GW182 gene family. GW localizes to punctate structures in the cytoplasm of Drosophila embryos and cultured S2 cells. Drosophila GWBs are electron-dense nonmembrane-bound cytoplasmic foci. These structures are targeted by human GW182 and its paralogues TNRC6B and TNRC6C in Drosophila cells. Unlike what is seen in some mammalian cells, only some foci colocalize with the previously identified GWB components LSm4, the Drosophila Xrn1 orthologue Pacman (PCM), and AGO2 (Ingelfinger, 2002; Eystathioy, 2003; Kedersha, 2005; Liu, 2005a; Sen, 2005). There is a requirement for the zygotic expression of full-length Drosophila GW during early embryonic nuclear divisions. This suggests a critical role for GWB-based cytoplasmic RNA regulation in Drosophila beginning with early embryo development (Schneider, 2006).

The results confirm that GW is homologous to human GW182 and that Drosophila GWBs are analogous to human GWBs and yeast PBs. GW localizes to rapidly moving and electron-dense, nonmembrane-bound cytoplasmic structures. Colocalization of GW to homologues of known GWB or PB components LSm4, AGO2, and PCM (Xrn1) shows that Drosophila GWBs are of similar composition to PBs and GWBs. Another similarity between GWBs and PBs is that Drosophila GWBs also require intact RNA to maintain their integrity. Functionally, human and Drosophila GW homologues are all targeted to the same foci when coexpressed in S2 cells. However, not all Drosophila GWBs contain the mRNA decay enzymes LSm4 and PCM or AGO2 associated with GWBs or PBs. There is an apparent lack of interdependence in functions of the nonsense-mediated decay, RNAi, and miRNA pathways in Drosophila S2 cells, as the depletion of proteins involved in one pathway did not affect the function of another (Rehwinkel, 2005). Thus, the variable composition of Drosophila GWBs provides evidence that there may be distinct functions for these cytoplasmic structures. It may be possible to discern functionally distinct classes of GWBs by analyzing relative localizations of other mRNA-processing proteins as they become known (Schneider, 2006).

There have been several exhaustive screens to identify zygotically transcribed genes that affect Drosophila precellular embryonic development. Currently, a total of seven genes are thought to be expressed before the cellular blastoderm stage. However, these screens focused on the X chromosome and autosomes two and three, but not four. It is proposed that gw represents an additional zygotically expressed gene required for successful completion of the early embryo development in Drosophila. The reduction in GW protein observed at 60-70 min AED suggests that maternally supplied GW is depleted. This would be subsequently replenished by zygotic gw transcription, as shown by rising mRNA levels beginning at 70-80 min AED, a time of rapid nuclear division that culminates in the cellularization and subsequent gastrulation steps of embryo development. Notably, increased levels of Drosophila GW expression are also observed during pupal development, which is another time of rapid cell proliferation. The increase in GW expression during periods of rapid cell division is consistent with elevated GW182 levels (Yang, 2004) observed in proliferating human cells (Schneider, 2006).

The function of GWBs described in mammalian cells suggests a potential role for these structures in Drosophila development. In many organisms, siRNA and miRNA, which are produced by Dicer-mediated cleavage of longer double-stranded or hairpin RNA precursors, regulate several developmental functions. For both siRNA and miRNA activity, the RNA-induced silencing complex (RISC) binds and selectively suppresses or degrades complementary target mRNA . Several recent studies have identified a link between GWBs and the RNAi pathway. RISC components Ago1-4 localize to GWBs (Liu, 2005b; Sen, 2005), as do reporter mRNAs targeted for miRNA-mediated translational repression (Liu, 2005b). In addition, intact GWBs are required for siRNA silencing (Jakymiw, 2005; Liu, 2005b). The effects of miRNA expression on Drosophila development were characterized in a screen of 46 embryonically expressed miRNAs. Injection of antisense RNA to block these miRNAs into 30-min AED embryos revealed 25 miRNAs with visible phenotypes affecting a variety of developmental processes. Blocking miR-9 resulted in several severe defects, including nuclear division and migration, actin cytoskeleton formation, and cellularization. A role for components of the RNAi machinery in the timing of heterochromatin formation and accurate chromosome separation has been reported in Schizosaccharomyces pombe and the trypanosome Trypanosoma brucei. Drosophila Ago2 mutants show several defects in early embryogenesis, including defects in centromeres, nuclear division, nuclear migration, and germ cell migration. However, homozygous Ago2 mutants are, for the most part, fertile and viable. Therefore, cytoplasmic-based RISC-mediated miRNA may have an effect on the control of timing of protein reorganization associated with cytoskeletal and mitotic events during early development (Schneider, 2006).

The putative C. elegans GW protein orthologue Ain-l localizes to cytoplasmic foci with a composition similar to PBs and GWBs and forms complexes with ALG-1 (argonaute-like gene) Dicer-1 and miRNAs. However, C. elegans Ain-1 and RNAi components dicer-1, alg-1, and alg-2 function in the heterochronic pathway that regulates developmental timing in many postembryonic cell lineages (Grishok, 2001; Ding, 2005), while xrn1 is required in embryogenesis (Newbury, 2004) for ventral epithelial closure (Schneider, 2006).

The phenotypes associated with blocking Drosophila GW function suggest that functional GWBs are required for the completion of nuclear divisions during early embryonic development. These effects, although similar to Drosophila Ago2 mutants, are far more severe. Injection of anti-AGO2 antibody into early embryos caused a reduction in number and enlargement in the size of the embryonic nuclei detected by NLS-GFP. The more severe defects resulting from GW depletion may be caused by the nature of the Ago2 mutation, which does not completely block protein function, or may be the consequence of additional functions of Drosophila GWBs (which are not related to AGO2) and, by extension, RISC function (Schneider, 2006).

Drosophila GW is expressed throughout development and is required for the viability of cultured Drosophila cells. The data suggest that one function of GWBs is to coordinate the regulation of embryonic development in a posttranscriptional fashion. Subsets of eukaryotic mRNAs involved in the same cellular processes are often associated with specific RNA-binding proteins, depending on growth conditions. In one proposed model, RNP particles like GWBs coordinately regulate mRNAs encoding functionally related proteins, which is analogous to the operon-based coordination of prokaryotic gene expression (Keene, 2005). Thus, mRNAs with similar cis-elements would be recognized and trafficked by a common RNP to collectively regulate their translation or degradation. These data provide evidence that Drosophila GWBs mirror human GWB composition and function, providing an excellent model for genetic dissection of the potential role of GWBs in regulating mRNAs during development (Schneider, 2006).

A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing: Regulation of silencing by Ago1

In eukaryotic cells degradation of bulk mRNA in the 5' to 3' direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5' to 3' exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mRNA decay (NMD), and the Argonaute proteins, which are essential for RNA interference (RNAi) and the micro-RNA (miRNA) pathway. In addition, XRN1 is required for degradation of mRNAs targeted by NMD and RNAi. To investigate a possible interplay between P-bodies and these post-transcriptional, processes P-body or essential pathway components were depleted from Drosophila cells and the effects of these depletions were analyzed on the expression of reporter constructs, allowing specific monitoring of NMD, RNAi, or miRNA function. The RNA-binding protein GW182 and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing, uncovering a crucial role for P-body components in the miRNA pathway. This analysis also revealed that inhibition of one pathway by depletion of its key effectors does not prevent the functioning of the other pathways, suggesting a lack of interdependence in Drosophila (Rehwinkel, 2005).

In eukaryotic cells, bulk messenger RNA (mRNA) is degraded via two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Following this first step, mRNAs can be degraded from their 3' ends by the exosome, a multimeric complex of 3' to 5' exonucleases. Alternatively, after deadenylation, the cap structure is removed by the DCP1:DCP2 decapping complex, and the mRNA is degraded by the major cytoplasmic 5' to 3' exonuclease XRN1 (Rehwinkel, 2005).

Proteins required for 5' to 3' mRNA degradation (e.g., DCP1, DCP2, and XRN1) colocalize in specialized cytoplasmic bodies or mRNA decay foci, also known as mRNA processing bodies (P-bodies) or GW-bodies, because of the accumulation of the RNA binding protein GW182 in these bodies. Additional components of P-bodies in yeast and/or human cells include the deadenylase Ccr4, the cap binding protein eIF4E and its binding partner eIF4E-transporter (eIF4E-T), auxiliary decay factors such as the LSm1-7 complex, Pat1p/Mtr1p, and the putative RNA helicase Dhh1/rck/p54. Among these, human GW182, eIF4E-T, and Dhh1 are required for P-body formation, while the decapping enzymes and XRN1 are dispensable. In addition, mRNA decay intermediates, microRNA (miRNA) targets, and miRNAs have been localized to P-bodies, suggesting that these bodies are sites where translationally silenced mRNAs are stored before undergoing decay (Rehwinkel, 2005 and references therein).

Recently, proteins involved in other post-transcriptional processes have been localized to P-bodies in human cells. These include the proteins SMG5, SMG7, and UPF1 involved in the nonsense-mediated mRNA decay (NMD) pathway and the Argonaute (AGO) proteins that play essential roles in RNA silencing. Moreover, XRN1 is recruited by both the NMD and the RNA interference (RNAi) machineries to degrade targeted mRNAs, suggesting a possible link between NMD, RNAi, and P-bodies. NMD is an mRNA quality control (or surveillance) mechanism that degrades aberrant mRNAs having premature translation termination codons (PTCs), thereby preventing the synthesis of truncated and potentially harmful proteins. Core components of the NMD machinery include the proteins UPF1, UPF2, and UPF3, which form a complex whose function in NMD is conserved. The activity of UPF1 is regulated in multicellular organisms by additional proteins (i.e., SMG1, SMG5, SMG6, and SMG7) that are also required for NMD in all organisms in which orthologs have been characterized (Rehwinkel, 2005 and references therein).

In yeast and human cells, a major decay pathway for NMD substrates involves decapping and 5' to 3' degradation by XRN1. Although degradation of nonsense transcripts in Drosophila is initiated by endonucleolytic cleavage near the PTC, the resulting 3' decay intermediate is also degraded by XRN1. A molecular link between the NMD machinery and the decay enzymes localized in P-bodies is provided by SMG7 in human cells. Indeed, when overexpressed, human SMG7 localizes in P-bodies and recruits both UPF1 and SMG5 to these bodies, suggesting that NMD factors may reside at least transiently in P-bodies. RNA silencing pathways are evolutionarily conserved mechanisms that elicit decay or translational repression of mRNAs selected on the basis of complementarity with small interfering RNAs (siRNAs) or miRNAs, respectively. siRNAs are fully complementary to their targets and elicit mRNA degradation via the RNAi pathway. Animal miRNAs are only partially complementary to their targets and do not generally elicit decay, but repress translation instead (Rehwinkel, 2005 and references therein).

To perform their function, the siRNAs and miRNAs associate with the AGO proteins to form multimeric RNA-induced silencing complexes (RISC). Drosophila AGO1 mediates miRNA function, while AGO2 catalyzes the endonucleoytic cleavage of siRNA targets within the region complementary to the siRNA. Following this initial cleavage, the resulting 5' mRNA fragment is degraded by the exosome, while the 3' fragment is degraded by XRN1. The localization of AGO proteins in P-bodies in human cells provides a possible link between these bodies and silencing pathways (Rehwinkel, 2005 and references therein).

The NMD, the siRNA, and the miRNA pathways are therefore interlinked by the use of common decay enzymes and/or the coexistence of components of these pathways in P-bodies, suggesting a possible interdependence between these post-transcriptional mechanisms. Evidence for a link between NMD and RNAi has been reported in Caenorhabditis elegans where UPF1, SMG5, and SMG6 are required for persistence of RNAi, though not to initiate silencing. In contrast, UPF2, UPF3, and SMG1, which are also essential for NMD, are not required to maintain silencing, suggesting that UPF1, SMG5, and SMG6 may have evolved specialized functions in RNAi (Rehwinkel, 2005 and references therein).

This study investigates the interplay between NMD, RNAi, and the miRNA pathway using the Drosophila Schneider cell line 2 (S2 cells) expressing reporters allowing the monitoring of NMD, RNAi, or miRNA function. To this end, factors involved in NMD (UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6), RNAi (AGO2), or the miRNA pathway (AGO1) were depleted and the effect on the expression of the reporters analyzed. These proteins showed a high degree of functional specificity. To determine the role of P-body components in these pathways the DCP1:DCP2 decapping complex, the decapping coactivators LSm1 and LSm3, the 5' to 3' exonuclease XRN1, GW182, and the Drosophila protein CG32016, which shares limited sequence homology with human eIF4E-T, were depleted. The results uncovered a crucial role for GW182 and the DCP1:DCP2 decapping complex in the miRNA pathway (Rehwinkel, 2005).

Components of the NMD, RNAi, and miRNA pathways exhibit functional specificity in Drosophila To investigate a potential role of components of RNA silencing pathways or of P-body components in NMD, use was made of cell lines expressing wild-type or PTC-containing reporter constructs in which the coding regions of the bacterial chloramphenicol acetyl transferase (CAT) or the Drosophila alcohol dehydrogenase (adh) genes were placed downstream of inducible or constitutive promoters. The PTCs were inserted at codon 72 and 83 of the CAT and adh open reading frames, respectively. P-body components and proteins involved in NMD, RNAi, or the miRNA pathway were depleted by treating the cells with double-stranded RNAs (dsRNAs) specific for the different factors. A dsRNA that targets green fluorescent protein (GFP) served as a control. The steady-state levels of the wild-type and PTC-containing mRNAs were analyzed by Northern blot and normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32) (Rehwinkel, 2005).

Relative to the expression levels of the wild-type mRNAs, the levels of the corresponding PTC-containing transcripts are reduced because these transcripts are rapidly degraded via the NMD pathway. Depletion of UPF1 inhibits NMD, so the levels of the PTC-containing mRNAs are restored. Depletion of AGO1 or AGO2, both singly and in combination, does not interfere with the NMD pathway, although these depletions do inhibit siRNA- or miRNA-mediated gene silencing. The levels of the CAT wild-type transcript were not affected by the depletions. Similar results were obtained with the NMD reporter based on the adh gene. Together, these results indicate that inhibition of RNAi or of the miRNA pathway does not interfere with NMD. XRN1 is the only P-body component known to be required for degradation of decay intermediates arising from mRNAs undergoing NMD in Drosophila. Nevertheless, in cells depleted of XRN1 the NMD pathway is not inhibited, and only the 3' decay intermediate generated by endonucleolytic cleavage of the mRNA accumulates (Rehwinkel, 2005).

In contrast to XRN1, none of the P-body components tested, including GW182 and the DCP1:DCP2 decapping complex, affected NMD or the accumulation of the 3' decay intermediate. The lack of a significant effect of the depletion of the DCP1:DCP2 complex was confirmed using the adh reporter. The decapping enzymes are certainly involved in NMD in yeast and human cells because the major decay pathway for NMD substrates is initiated by decapping in these organisms (for review, see Conti, 2005). Thus, it is possible that the requirement for P-body components and/or P-body integrity in NMD varies across species (Rehwinkel, 2005).

Two different approaches were used to investigate the RNAi pathway. In one approach, a cell line constitutively expressing the wild-type Drosophila adh gene was treated with a dsRNA complementary to a central region of ~300 nucleotides (nt) of adh mRNA (adh dsRNA). This dsRNA elicits decay of the adh mRNA via the RNAi pathway. Cells were treated with dsRNAs targeting various factors in the presence or absence of adh dsRNA. The steady-state levels of the adh mRNA were analyzed by Northern blot and normalized to those of the rp49 mRNA. In cells treated with GFP dsRNA, the normalized levels of the adh transcript were reduced to 4% after addition of adh dsRNA, relative to the levels detected in the absence of adh dsRNA. In cells depleted of AGO2, a sixfold increase of adh mRNA levels was observed despite the presence of adh dsRNA. In contrast, when AGO1 was depleted, adh dsRNA could still trigger a reduction of adh mRNA levels, though a slight increase in transcript levels was observed. Similarly, depletion of UPF1 did not prevent silencing of adh expression by adh dsRNA. These results indicate that UPF1 is not required for RNAi in Drosophila. Additional NMD components (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6) have been identified, but no SMG7 ortholog has been identified in Drosophila. No significant change was observed in the efficacy of RNAi under the conditions in which NMD was inhibited (Rehwinkel, 2005).

Similarly to the results reported for the NMD pathway, depletion of XRN1 leads to the accumulation of the 3' decay intermediate generated by endonucleolytic cleavage by RISC, while depletion of the DCP1:DCP2 decapping complex does not prevent RNAi or the degradation the 3' decay intermediate. In contrast, depletion of GW182 leads to a modest increase in the adh mRNA level in the presence of adh dsRNA, suggesting that this protein could influence the efficiency of RNAi (Rehwinkel, 2005).

In a second approach, RNAi was triggered by an siRNA instead of a long dsRNA, to uncouple RISC activity from processing of dsRNAs. To this end, S2 cells were transiently transfected with a plasmid expressing firefly luciferase (F-Luc) and an siRNA targeting the luciferase coding sequence (F-Luc siRNA) or a control siRNA. A plasmid encoding Renilla luciferase (RLuc) was included to normalize for transfection efficiencies. Cotransfection of the F-Luc reporter with the F-Luc siRNA led to a 50-fold inhibition of firefly luciferase activity relative to the activity measured when the control siRNA was cotransfected, indicating that F-Luc siRNA effectively silences firefly luciferase expression (Rehwinkel, 2005).

The results obtained with the luciferase reporter correlate well with those obtained with adh mRNA, in spite of differences between the methods used to detect changes in reporter levels (RNA levels vs. protein levels), and the nature of the RNA trigger (long dsRNA vs. siRNA). Indeed, depletion of AGO2 impaired silencing of firefly luciferase expression by the F-Luc siRNA, leading to an eightfold increase in firefly luciferase activity relative to the activity of the Renilla control. Depletion of AGO1 led to a twofold increase of firefly luciferase activity (Rehwinkel, 2005).

The observation that depletion of AGO2, but not AGO1, significantly inhibits RNAi is in agreement with previous reports showing that only AGO2-containing RISC is able to catalyze mRNA cleavage triggered by siRNAs. The results together with these observations indicate that Drosophila AGO1 and AGO2 are not redundant (Rehwinkel, 2005).

Depletion of GW182 or the DCP1:DCP2 complex led to a 1.5- to twofold increase of the firefly luciferase activity, although RNAi was not abolished. These results together with those obtained with the adh reporter suggest that GW182 and the DCP1:DCP2 complex are not absolutely required for RNAi but may modulate siRNA function (Rehwinkel, 2005).

Finally, depletion of core NMD components does not inhibit the silencing of firefly luciferase expression by F-Luc siRNA. The results are consistent with results from C. elegans showing that NMD per se is not required for the establishment of silencing (Rehwinkel, 2005).

To investigate the miRNA pathway firefly luciferase reporters were generated in which the coding region of firefly luciferase is flanked by the 3' UTRs of the Drosophila genes CG10011 or Vha68-1. These genes were identified as miRNA targets in a genome-wide analysis of mRNAs regulated by AGO1. The 3' UTR of CG10011 mRNA contains two binding sites for miR-12, while the 3’ UTR of Vha68-1 has two binding sites for miR-9b. Expression of the firefly luciferase construct fused to the 3' UTR of CG10011 (F-Luc-CG10011) was strongly reduced by cotransfection of a plasmid expressing the primary (pri) miR-12 transcript, but not pri-miR-9. Conversely, expression of the firefly luciferase reporter fused to the 3' UTR of Vha68-1 (FLuc-Vha68-1) was inhibited by cotransfection of pri-miR-9b, but not of primiR-12 (Rehwinkel, 2005).

Silencing of luciferase expression by the cognate miRNAs was prevented in cells depleted of AGO1. Indeed, despite the presence of the transfected miRNAs, in cells depleted of AGO1 an 11-fold and a 16-fold increase of firefly luciferase expression was observed from the FLuc- CG10011 and F-Luc-Vha68-1 reporters, respectively. Notably, the firefly luciferase activity measured in AGO1-depleted cells in the presence of the transfected miRNAs was at least twofold higher than the activity measured in control cells in the absence of exogenously added miRNAs. Since endogenous miR-9b and miR-12 are expressed in S2 cells, these results suggest that depletion of AGO1 also suppresses silencing mediated by the endogenous miRNAs. Depletion of AGO2 does not suppress the effect of coexpressing the reporters with the cognate miRNAs. These results provide additional evidence supporting the conclusion that the siRNA and miRNA pathways are not interdependent (Rehwinkel, 2005).

miRNA-mediated silencing of firefly luciferase expression was not affected by depletion of UPF1 or by the additional NMD factors (i.e., UPF2, UPF3, SMG1, SMG5, and SMG6). Thus, the individual NMD factors and NMD per se are not required for miRNA function. Unexpectedly, although the efficiency of NMD and RNAi was unaffected or only modestly affected in cells depleted of GW182 or the DCP1:DCP2 complex, miRNA-mediated silencing of firefly luciferase expression was effectively relieved in these cells. In the presence of cognate miRNAs, depletion of GW182 resulted in a sixfold increase of firefly luciferase expression. Therefore, despite the presence of transfected miRNAs, firefly luciferase activity in GW182-depleted cells was similar to that measured in controls cells in the absence of transfected miRNAs. Codepletion of DCP1 and DCP2 led to a fourfold increase of firefly luciferase expression. Finally, depletion of CG32016 resulted in a twofold increase of firefy luciferase activity, but only for the F-Luc-Vha68-1 reporter, suggesting that this effect may not be significant (Rehwinkel, 2005).

To investigate whether depletion of GW182 affects RISC activity directly, as opposed to interfering with miRNA processing, use was made of a tethering assay. This assay involves the expression of a lN-fusion of AGO1 that binds with high affinity to five BoxB sites (5-BoxB) in the 3’ UTR of a firefly luciferase reporter mRNA. When AGO1 is tethered to this reporter transcript, luciferase expression is inhibited relative to the activity measured in cells expressing the lN-peptide alone. The inhibition was partially relieved in cells depleted of GW182 but not of AGO2. It is concluded that GW182 and the decapping DCP1: DCP2 complex play a critical role in the effector step of the miRNA pathway. These results are in agreement with the observation that Argonaute proteins localize to P-bodies and interact with DCP1 and DCP2 independently of RNA or of P-body integrity (Rehwinkel, 2005).

Thus, despite convergence in P-bodies, NMD, RNAi, and the miRNA pathway are not interdependent in Drosophila. This conclusion is based on the observation that the inhibition of one pathway by depleting key effectors may slightly interfere with, but does not significantly inhibit, the functioning of the other pathways. The lack of interdependence between RNAi and the miRNA pathway is further supported by the observation that knockouts of AGO1 or AGO2 in Drosophila have different phenotypes. Nevertheless, cross-talk between the RNAi and the miRNA pathways may still occur at the initiation step, since Dicer-1 plays a role in RISC assembly (Rehwinkel, 2005).

Biochemical and genetic approaches in several organisms have led to the identification of essential components of the miRNA pathway. These include AGO1 and the enzymes required for miRNA processing, such as Drosha and Dicer-1 and their respective cofactors, Pasha and Loqs. However, the mechanisms by which miRNAs inhibit protein expression without affecting mRNA levels are not completely understood. Recent evidence suggests that translation initiation is inhibited and that the targeted mRNAs are stored in P-bodies, where they are maintained in a silenced state either by associating with proteins that prevent translation or possibly by removal of the cap structure. This study identified the P-body components GW182 and the DCP1:DCP2 decapping complex as proteins required for the miRNA pathway. The precise molecular mechanism by which these proteins participate in this pathway remains to be established. These proteins may have an indirect role in the miRNA pathway by affecting P-body integrity. Alternatively, these proteins may play a direct role in this pathway by escorting miRNA targets to P-bodies or facilitating mRNP remodeling steps required for the silencing of these targets. Consistent with a direct role for the DCP1:DCP2 decapping complex, and thus for the cap structure, in miRNA function is the observation that mRNAs translated via a cap-independent mechanism are not subject to miRNA-mediated silencing. In conclusion, the results uncover an important role for the P-body components, GW182 and the DCP1:DCP2 complex, in miRNA-mediated gene silencing (Rehwinkel, 2005).

GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets

miRNAs are posttranscriptional regulators of gene expression that associate with Argonaute and GW182 (Drosophila Gawky) proteins to repress translation and/or promote mRNA degradation. miRNA-mediated mRNA degradation is initiated by deadenylation, although it is not known whether deadenylases are recruited to the mRNA target directly or by default, as a consequence of a translational block. To answer this question, a screen was performed for potential interactions between the Argonaute and GW182 proteins and subunits of the two cytoplasmic deadenylase complexes. Human GW182 proteins were found to recruit the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complexes through direct interactions with PAN3 and NOT1, respectively. These interactions are critical for silencing and are conserved in D. melanogaster. These findings reveal that GW182 proteins provide a docking platform through which deadenylase complexes gain access to the poly(A) tail of miRNA targets to promote their deadenylation, and they further indicate that deadenylation is a direct effect of miRNA regulation (Braun, 2011; graphic abstract of article).

Emerging evidence suggests that mRNA deadenylation is part of the mechanism used by miRNAs to silence gene expression. Indeed, deadenylation of miRNA targets has now been reported in zebrafish and C. elegans embryos, human and D. melanogaster cells, and in various cell-free extracts that recapitulate silencing. However, whether miRISCs directly recruit deadenylases to miRNA targets has remained unclear (Braun, 2011).

This study provides compelling evidence that the silencing domains (SDs) of TNRC6 proteins (human GW182 paralogs) contain binding sites for PAN3 and NOT1, which are subunits of each of the two major cytoplasmic deadenylase complexes. These findings provide strong support for the hypothesis that GW182 proteins enhance poly(A) tail removal by directly recruiting deadenylases to associated mRNA targets. More broadly, these results have implications for the understanding of miRNA-based regulation, because they show that target deadenylation is not merely a consequence of a translational block (Braun, 2011).

Previous studies have reported conflicting evidence regarding the interaction of deadenylation factors with the two major components of miRISCs (AGO and GW182). Indeed, several studies failed to detect a significant interaction between human AGO or TNRC6 proteins and components of deadenylase complexes, including POP2, CAF1, CCR4a, CCR4b, and PAN2 (Braun, 2011).

Using coimunoprecipitation and in vitro pull-down assays, it was determined that PAN3 and NOT1 interact directly with TNRC6-SDs, whereas the interaction with PAN2 and the additional components of the CCR4-CAF1-NOT complex is indirect and bridged by PAN3 and NOT1, respectively. These observations provide one explanation for the negative results reported in previous studies. Indeed, other studies focused on the interaction of AGO and GW182 with subunits of the deadenylase complexes that interact indirectly (e.g., the catalytic subunits and NOT3). These indirect interactions are likely to be affected by the efficiency of the immunoprecipitation and the expression of the tagged proteins relative to the expression of the endogenous bridging factors. In agreement with this interpretation, this study showed that human TNRC6C did not coimmunoprecipitate PAN2; nevertheless, an interaction with PAN2 was observed when PAN3 (the bridging factor) was overexpressed (Braun, 2011).

Previous studies have shown that the silencing domain of GW182 proteins contains two binding sites for PABPC1: one in the PAM2 motif and one in the M2 and C-terminal regions. The PAM2 motif interacts directly with the C-terminal MLLE domain of PABPC1. The M2 and C-terminal regions mediate indirect binding to PABPC1, which is only observed in cell lysates. This study has shown that the TNRC6 M2 and C-term regions mediate direct binding to PAN3. PAN3, in turn, binds to PABPC1 and PAN2 and may act as a bridging factor. It was also shown that the M1, M2, and C-term regions of the silencing domain confer direct binding to NOT1, which, in turn, mediates interaction with the additional subunits of the CCR4-CAF1-NOT complex (Braun, 2011).

A model is presented that summarizes the interactions uncovered in this work as well as those from previous studies. TNRC6 proteins are recruited to miRNA targets through their interaction with AGOs, and they contact PABPC1 directly through their PAM2 motifs. TNRC6 proteins also bind PAN3 and NOT1 via their Mid and C-term regions, as shown in this study. These interactions may occur consecutively, simultaneously, or alternatively. PAN3 interacts with the catalytic subunit PAN2 . Additionally, PAN3 contains an N-terminal PAM2 motif that could bind to the MLLE domain of a second PABPC1 molecule. Finally, NOT1 recruits the additional subunits of the CCR4-CAF1-NOT complex. Although the detailed molecular interactions between the deadenylases, PABPC1 and TNRC6s need to be further elucidated, an important conclusion emerging from these studies is that TNRC6 proteins engage in multiple interactions with deadenylases and PABPC1 to promote target mRNA degradation. Moreover, the observation that depletion of PAN3 and NOT1 suppresses silencing of an unadenylated reporter, suggests that deadenylase complexes could also contribute to translational repression in addition to promoting deadenylation and decay. Thus, it is possible that translational repression and deadenylation are two distinct outcomes triggered by the recruitment of deadenylase complexes to the 3′UTR of miRNA targets. Further studies will determine how deadenylase complexes interact with TNRC6 proteins at the molecular level, and the role they may play in translational repression (Braun, 2011).

PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding

Polyadenylated mRNAs are typically more strongly repressed by microRNAs (miRNAs) than their nonadenylated counterparts. Using a Drosophila melanogaster cell-free translation system, this effect was found to be mediated by the poly(A)-binding protein (PABP). miRNA repression was positively correlated with poly(A) tail length, but this stimulatory effect on repression was lost when translation was repressed by the tethered GW182 silencing domain rather than the miRNA-induced silencing complex (miRISC) itself. These findings are mechanistically explained by a notable function of PABP: it promotes association of miRISC with miRNA-regulated mRNAs. It was also found that PABP association with mRNA rapidly diminished with miRISC recruitment and before detectable deadenylation. These data were integrated into a revised model for the function of PABP and the poly(A) tail in miRNA-mediated translational repression (Moretti, 2012).

Although the poly(A) tail and PABP are important effectors of miRNA function in many but not all experimental systems, the molecular mechanism(s) by which they contribute to miRNA-mediated silencing is unclear. This study used a combination of biochemical and functional assays in the D. melanogaster embryo in vitro system to discover a new aspect of this interplay and to show that stimulation of miRNA-mediated repression by the presence of a poly(A) tail is mediated by PABP (Moretti, 2012).

miRISC has been proposed to interfere with the interaction of PABP with eIF4G, causing a disruption of closed-loop formation and translational repression. The data reported in this study show a role of PABP in miRNA-mediated repression. It was found that the length of the poly(A) tail (and therefore the number of PABP molecules that it can accommodate) is positively correlated with the extent of miRNA-mediated repression. Analogously, for a reporter mRNA bearing only a single miRNA-binding site, the presence of the poly(A) tail is essential to confer repressibility; this reporter is not susceptible to repression in its nonadenylated form. Furthermore, the extent of repression of reporter mRNAs controlled by GW182-SD tethering is not influenced by the presence or absence of a poly(A) tail, although the poly(A) tail stimulates translation of these reporters; this result uncouples translatability and repressibility. Efficient repression by GW182 protein tethering to reporters lacking a poly(A) tail has also been observed with reporters bearing either a histone stem-loop structure or a hammerhead ribozyme cleavage site at their 3′ ends. These data all suggest that a physiological miRISC context is required for PABP and the poly(A) tail to stimulate repression (Moretti, 2012).

Deadenylation of target mRNAs is a widespread effect of miRNA-mediated repression and involves PABP. Several studies have suggested that PABP is displaced from the mRNA before deadenylation starts and proposed that PABP affinity for the poly(A) tail is modified either by specific domains of PABP itself or by mRNA-specific activators of deadenylation. In agreement with these studies, this study shows that miRNA-mediated repression (and repression achieved through GW182-SD tethering) causes lower target mRNA association with PABP. This observation supports the idea that the PABP-miRISC interaction could reduce the affinity of PABP for the poly(A) tail, rendering it more accessible to deadenylase. These experiments show that PABP dissociates from mRNAs in parallel with AGO1 association early in the establishment of silencing and notably before deadenylation begins. Therefore, PABP dissociation is not a mere consequence of deadenylation, and deadenylation could be facilitated by the loss of PABP. GW182 proteins can directly recruit components of deadenylase complexes (Braun, 2011; Chekulaeva, 2011; Fabian, 2011). Taken together, these observations support the notion that miRISC coordinates both PABP displacement and recruitment of the deadenylation machinery to efficiently promote deadenylation of miRNA targets (Moretti, 2012).

The above results also offer a mechanistic explanation for how PABP and the poly(A) tail stimulate miRISC action: they exert their stimulatory function, at least in part, by facilitating miRISC binding to target mRNAs. Does PABP facilitate miRISC binding by enhanced recruitment or by stabilization of the miRISC complex on a target mRNA? Without direct measurement of on and off rates of the miRISC complex it is not possible todiscriminate between these two possibilities. However, the first alternative is favored: if PABP stabilized miRISC on the mRNA over time, it would be expected to remain associated with the repressed mRNP to exert its effect. In contrast with this prediction, it was found that PABP dissociates rather early. If PABP supports miRISC recruitment, mRNAs that are already affected by miRNA-mediated repression would have been deadenylated, bear markedly lower amounts of PABP and therefore compete less efficiently for miRISC. Conversely, mRNAs that still have to be repressed will bear longer poly(A) tails and their associated PABP will promote miRISC recruitment. This effect would provide a mechanism for efficiently targeting miRISC complexes to mRNAs that should undergo repression. The RNA-binding protein HuR has also been proposed to stimulate miRNA-mediated repression by facilitating miRISC recruitment to target mRNAs. As GW182 mutants that do not interact with PABP are severely impaired in silencing (Huntzinger, 2010; Braun, 2011), it is speculated that PABP facilitates miRISC recruitment through its direct interaction with GW182, although bridging binding partners could also explain the data. PABP has been proposed to be dispensable for miRNA-mediated repression in vitro. Although the data support the notion that PABP is not absolutely required for miRNA-mediated repression in vitro, this study demonstrates an important function of PABP in silencing. The in vitro system referred to above relies on preloading of reporter-specific miRISC with exogenously supplemented miRNA duplexes. It is predicted that such an experimental system misses the function of PABP that is described in this paper (Moretti, 2012).

Notably, the stimulatory effect of the poly(A) tail on AGO1 association was more pronounced for reporter mRNAs bearing 3′ UTRs of sickle (skl) or grim. Although additional miRNAs might bind skl and grim 3′ UTRs, this study observed a marked reduction (three- to five-fold) of AGO1 association upon incubation with anti-miR2 LNAs, indicating the importance of the miR2-RISC. Notably, the miR2-binding sites within these 3′ UTRs have a less favorable pairing with miR2 than the reaper 3′ UTR-binding motif present in the 1× and 6× reporters as indicated by the Z-score, an indicator of the folding energy of each miRNA-mRNA target pair. This inverse correlation suggests that the more pronounced stimulatory effect of PABP and the poly(A) tail on miRISC association results from the less favorable Watson-Crick base pairing. Potentially, the poly(A) tail and PABP contribute less to miRISC association with high-affinity binding sites showing strong Watson-Crick base pairing (Moretti, 2012).

On the basis of this data, an extended model is proposed for miRNA-mediated silencing in which PABP and the poly(A) tail augment binding of miRISC to the target mRNA during early phases of silencing. miRISC binding induces displacement of PABP and, upon recruitment of deadenylase complexes, deadenylation of target mRNA. This model does not challenge but rather extends earlier analyses of the functional importance of miRISC-PABP interaction and clarifies the complex interplay among miRISC, the translation initiation machinery and establishment of effective silencing (Moretti, 2012).

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. This study demonstrates that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. These findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation (Zekri, 2013).

miRISC recruits decapping factors to miRNA targets to enhance their degradation

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. This study, carried out in Drosophila S2 cells, demonstrates that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation (Nishihara, 2013).

This study demonstrates that miRISCs enhance the association of DCP1, Me31B and HPat with miRNA targets in a miRNA-dependent manner. This association occurs even when the miRNA target lacks a 5' cap structure, an ORF and a poly(A) tail. Furthermore, mRNA reporters that are immune to deadenylation are degraded through decapping in the presence of the miRNA, indicating that miRISCs can promote decapping independently of deadenylation (Nishihara, 2013).

It is known that miRNAs promote the degradation of partially complementary targets through the 5'-to-3' decay pathway. In this pathway, decapping is coupled to deadenylation and does not occur on polyadenylated and fully functional mRNAs. This study investigated whether the decapping of miRNA targets occurs by default, as a consequence of this coupling, or whether miRISCs can also recruit decapping factors independently of deadenylation. miRISCs was shown to enhance the association of DCP1, Me31B and HPat with unadenylated 7SL-derived miRNA targets that have been transcribed by Pol III, indicating that the cap, a poly(A) tail and ongoing deadenylation are not required for the recruitment of decapping factors to miRNA targets. DCP1 association with the Alu-miRNA target reporterers, termed EvAluator reporters, was strictly miRNA dependent and stimulated by GW182. miRNAs and GW182 also stimulated the association of HPat and Me13B with the EvAluator reporters, indicating that these decapping factors interact with miRISC components that are bound to EvAluator RNA. However, DCP1 and Me31B did not interact with isolated AGO1 or GW182 in co-immunoprecipitation assays, suggesting that the interaction of decapping factors with miRISC is indirect or that DCP1 and Me31B recognize AGO1 and GW182 as a complex. Indeed, it is possible that the decapping factors are recruited by the PAN2-PAN3 or CCR4-NOT deadenylase complexes, which interact with GW182 proteins directly. Alternatively, DCP1 and Me31B might recognize AGO1 or GW182 only in a certain conformation that is adopted on target binding. Although HPat did interact with AGO1 and GW182 in co-immunoprecipitation assays, these interactions were apparently not sufficient to enhance the association of HPat and a polyadenylated miRNA target. Nevertheless, it is possible that these interactions contribute to the recruitment of HPat to deadenylated or oligoadenylated targets (Nishihara, 2013).

A previous study in human cells reported that EDC4 co-localized with a specific miRNA target in a miRNA-dependent manner, whereas DCP1 and RCK (the human ortholog of Dm Me31B) associated with the target, regardless of the presence of the miRNA. In agreement with that study, this study observed that decapping factors associate with miRNA targets in the absence of the miRNA; however, it was found that their binding is enhanced by the cognate miRNA. This enhancement was observed for targets that are not degraded or when degradation of the target was partially inhibited and may have escaped detection in co-localization studies (Nishihara, 2013).

A functional implication for the association of decapping factors with miRNA-targets is that miRNA targets can be decapped and degraded even in the absence of a poly(A) tail or ongoing deadenylation. In combination with previously published data, the current results suggest that miRISC has multiple and redundant activities to ensure robust gene regulation: it induces translational repression, deadenylation and decapping, the latter in both a deadenylation-dependent and -independent manner (Nishihara, 2013).

Under which circumstances can deadenylation-independent decapping contribute to silencing? Decapping might play a role in silencing specific miRNA targets when deadenylation is blocked or when decapping is blocked and targets that have undergone deadenylation accumulate. Indeed, deadenylation and decapping can be uncoupled on specific mRNAs, in different cell types and under various cellular conditions, leading to the accumulation of deadenylated repressed mRNAs. These mRNAs can re-enter the translational pool on polyadenylation or might be degraded in a deadenylation-independent manner once decapping resumes. For example, in immature mouse oocytes, DCP2 and DCP1 are not detectable, but their expression increases during oocyte maturation. Consequently, in immature oocytes, many maternal mRNAs (most likely including miRNA targets) accumulate in a deadenylated silenced form. These mRNAs may be polyadenylated and translated at later stages of oogenesis or embryogenesis. However, a fraction of these deadenylated targets may be degraded through decapping when DCP2 and DCP1 are expressed. Additionally, DCP1 and DCP2 are phosphorylated under cellular stress conditions, and DCP1 is hyperphosphorylated during mitosis. Under these conditions, a subset of mRNAs is stabilized, suggesting that DCP1 and DCP2 phosphorylation inhibits decapping. Thus, it is possible that under various stress conditions, miRNA targets accumulate in a deadenylated form because decapping is inhibited and that deadenylation-independent decapping is required for the clearance of these targets on return to normal cellular conditions (Nishihara, 2013).

Notably, in addition to their role in target degradation, decapping activators act as general repressors of translation even in the absence of decapping. Therefore, these factors could play a more direct role in the translational repression of miRNA targets in the absence of mRNA degradation (Nishihara, 2013).

In contrast to translational repression and deadenylation, decapping irreversibly shuts down translation initiation and commits mRNA to full degradation. Thus, decapping prevents the reversal of miRNA-mediated silencing. However, some miRNA targets have been shown to be released from miRNA-mediated repression in response to extracellular signals, suggesting that decapping is somehow blocked for these targets to allow for a fast reversal of their repression. How decapping is prevented in a target-specific manner remains unclear, but it can reasonable be expected that proteins associated with these targets block decapping in cis by preventing DCP2 access to the cap structure. These proteins may bind the cap structure directly or may act indirectly, for example, by stabilizing binding of the cap-binding protein eIF4E to the mRNA. Proteins that act as inhibitors of DCP2-mediated decapping have been described and include Variable Charged X chromosome VCX-A protein, YB-1, Y14 and Dm CUP. Thus, it is possible that additional proteins that prevent the decapping of specific mRNAs are present in eukaryotic cells. Such mRNA-specific decapping regulators would be likely to play an important role in controlling the reversibility of silencing. Alternatively, mRNAs can be recapped in the cytoplasm; however, how recapping is regulated remains unknown (Nishihara, 2013).

In addition to the aforementioned sequence-specific decapping regulators, the cap-binding protein eIF4E acts as a general inhibitor of decapping by limiting DCP2 access to the cap structure. Therefore, for decapping to occur, eIF4E needs to dissociate from the 5' end of the mRNA. This study shows that eIF4E remains bound to at least a fraction of silenced miRNA targets in cells in which decapping is blocked. Furthermore, the DCP2 catalytic mutant did not detectably associate with the mRNA target, even though its overexpression inhibited decapping. These observations suggest that DCP2 does not stably associate with miRNA targets. Similarly, DCP2 did not co-localize with miRNA targets in human cells, although in these cells, EDC4 co-localized with the target in a miRNA-dependent manner. Thus, the process of decapping may involve multiple consecutive steps, including the association of decapping activators with the target mRNA in the absence of DCP2, eIF4E dissociation, DCP2 recruitment and cap hydrolysis. The current results suggest that miRISC facilitates an early decapping step by increasing the local concentration of decapping factors on mRNA targets, promoting decapping independently of deadenylation. Further studies are necessary to determine whether, in addition to recruiting decapping factors, miRISC plays a more direct role in accelerating the chemical catalysis step of decapping (Nishihara, 2013).

MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. Previously studies have shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. This study examined the assembly of translation initiation factors on the target mRNA under repression. Ago1-RISC was shown to induce dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block the assembly of the eIF4F complex during translation initiation (Fukaya, 2014).

MicroRNAs (miRNAs) silence their complementary target mRNAs via formation of the effector ribonucleoprotein complex called RNA-induced silencing complex (RISC). The core component of RISC is a member of the Argonaute (Ago) proteins. In Drosophila, miRNAs are sorted into two functionally distinct Ago proteins, Ago1 and Ago2, according to their structural features and the identity of the 5' end nucleotides. Compared to fly Ago2, fly Ago1 shares more common features with mammalian Ago1-4, making it a suitable model for investigating miRNA-mediated gene silencing in animals. Ago1-RISC mediates translational repression as well as shortening of the poly(A) tail followed by mRNA decay (Behm-Ansmant, 2006). While deadenylation per se disrupts the closed-loop configuration of mRNA and leads to inhibition of translation initiation, Ago1-RISC can repress translation independently of deadenylation (Fukaya and Tomari, 2011). Such a deadenylation-independent 'pure' translational repression mechanism seems to be widely conserved among species (Bazzini, 2012, Bethune, 2012, Mishima, 2012 and Iwakawa and Tomari, 2013; Fukaya, 2014 and references therein).

Ago is not the only protein involved in the miRNA-mediated gene silencing pathway. In flies, a P-body protein GW182 specifically interacts with Ago1, but not with Ago2, through the N-terminal glycine/tryptophan (GW) repeats and provides a binding platform for PAN2-PAN3 and CCR4-NOT deadenylase complexes (Braun, 2011; Chekulaeva, 2011). This protein interaction network is conserved in animals including zebrafish, nematodes, and humans (Fabian, 2011; Kuzuoglu-Ozturk, 2012; Mishima, 2012). Accordingly, GW182 is essential for shortening of the poly(A) tail by miRNAs. In contrast, recent studies revealed that miRNA-mediated translational repression occurs in both GW182-dependent and -independent manners (Fukaya, 2012; Wu, 2013). Previous sedimentation analysis on sucrose density gradient suggested that both of the two translational repression mechanisms block recruitment of the ribosomal 43S preinitiation complex to the target mRNA independently of deadenylation (Fukaya, 2012; Fukaya, 2014 and references therein).

In eukaryotes, recruitment of the 43S preinitiation complex is initiated by the formation of eukaryotic translation initiation factor 4F (eIF4F). eIF4F is a multiprotein complex composed of the cap-binding protein eIF4E, which recognizes the 7-methyl guanosine (m7G) structure of the capped mRNA; the scaffold protein eIF4G, which interacts with 40S ribosome-associated eIF3 and bridges the mRNA and the 43S preinitiation complex; and the DEAD-box RNA helicase eIF4A, which plays a pivotal role in translation initiation supposedly through unwinding the secondary structure of the 5' UTR for landing of the 43S complex. In addition, the poly(A)-binding protein PABP stimulates translation initiation through its direct interaction with eIF4G. miRNAs likely block one (or more) of these steps to repress translation initiation. It was recently proposed that, in mammals, preferential recruitment of eIF4AII (one of the two eIF4A paralogs) is required for miRNA-mediated translational repression (Meijer, 2013). This model postulates that eIF4AII acts to inhibit rather than activate translation, unlike its major counterpart eIF4AI. However, the role of eIF4AII in translation remains largely unexplored, as opposed to eIF4AI's well-established function to promote translation. Moreover, invertebrates have only one eIF4A, making this model incompatible in flies. Thus, it still remains unclear how miRNAs repress translation initiation. This is largely due to technical limitations in directly monitoring the assembly of the translation initiation complex specifically on the mRNA targeted by miRNAs (Fukaya, 2014).

Using site-specific UV crosslinking this study examined the association of translation initiation factors on the target RNA under repression. Fly Ago1-RISC specifically induces dissociation of eIF4A from the target mRNA without affecting the 5' cap recognition by eIF4E in a manner independent of GW182 or PABP. On the other hand, direct tethering of GW182 to the target mRNA promotes dissociation of both eIF4E and eIF4A. It is proposed that miRNAs act to block assembly of the eIF4F complex during translation initiation, in addition to their established role in deadenylation and decay of their target mRNAs (Fukaya, 2014).

Although eIF4G could not be detected via any of the crosslinking positions spanning from 2 nt to 13 nt downstream of the cap, previous studies have shown that noncanonical translation driven by direct tethering of eIF4G to the 5' UTR was fully susceptible to translational repression by Ago1-RISC (Fukaya, 2012). Therefore, it was reasoned that Ago1-RISC directly targets eIF4A rather than eIF4E or eIF4G. In the accompanying paper, Fukao (2014) revealed that human Ago2-RISC specifically induces dissociation of eIF4A-both eIF4AI and eIF4AII-without affecting eIF4E or eIF4G in a cell-free system deriving from HEK293F cells (Fukao, 2014). Thus, eIF4A is likely a target of miRNA action conserved among species. In agreement with this model, miRNA-mediated gene silencing is cancelled by the eIF4A inhibitors silvestrol (Fukao, 2014), hippuristanol, or pateamine A (Leung, 2011; Meijer, 2013) in human cells (Fukaya, 2014).

GW182 is a well-known interactor of miRNA-associated Ago proteins and is a prerequisite for miRNA-mediated deadenylation/decay of target mRNAs (Behm-Ansmant, 2006). GW182 directly binds to both NOT1 and CAF40/CNOT9, thereby recruiting the CCR4-NOT deadenylase complex to the target mRNA. It has been suggested that the CCR4-NOT complex not only shortens the poly(A) tail but also plays a role in miRNA-mediated translational repression, because direct tethering of the CCR4-NOT complex was capable of inducing translational repression independently of deadenylation. It was originally proposed that, in humans, the CCR4-NOT complex specifically binds to eIF4AII (but not to eIF4AI) to repress translation. However, this model was challenged by recent studies showing that, although the MIFG4 domain of human CNOT1 structurally resembles the middle domain of eIF4G, it does not bind eIF4AI or II but instead partners with the DEAD-box RNA helicase DDX6, which has been implicated in repression of translation initiation and/or translation elongation as well as activation of decapping. Given that miRNAs mediate gene silencing via multiple different pathways, recruitment of DDX6 by GW182 via the CCR4-NOT complex may well play a role in inhibiting protein synthesis from miRNA targets. Indeed, this study observed strong dissociation of both eIF4E and eIF4A by direct tethering of GW182. However, at the physiological stoichiometry between Ago1 and GW182 in S2 cell lysate, eIF4A was specifically dissociated without apparent effect on eIF4E by canonical miRNA targeting, which is in agreement with the result of the reporter assay in S2 cells depleted of each eIF4F component. It is envisioned that, although GW182 is clearly essential for miRNA-mediated deadenylation, the degree of contribution of GW182 to translational repression can vary in different cell types and conditions, depending on the concentrations of GW182 and Ago proteins, as well as their protein interaction networks that are subject to regulation by extracellular signaling. In this regard, direct tethering of GW182 may potentially overestimate its role in miRNA-mediated translational repression (Fukaya, 2014).

How could Ago1-RISC specifically dissociate eIF4A from the initiation complex? Previous work has shown that none of GW182, the CCR4-NOT complex, or PABP is required for translational repression by Ago1-RISC (Fukaya, 2012). The current data extend these findings to reveal that Ago1-RISC can induce dissociation of eIF4A independently of GW182 or PABP. It is tempting to speculate that an as-yet-unidentified factor associated with Ago1-RISC, or perhaps Ago1-RISC itself, blocks the interaction between eIF4G and eIF4A (e.g., similarly to Programmed Cell Death 4 [PDCD4] whose tandem MA-3 domains compete with the MA-3 domain of eIF4G to bind the N-terminal domain of eIF4A, thereby displacing eIF4A from the eIF4F initiation complex). Alternatively, Ago1-RISC might directly or indirectly inhibit the ATP-dependent RNA-binding activity of eIF4A, which is tightly regulated by its accessory proteins eIF4B and eIF4H (Abramson, 1988; Richter, 1999). Future studies are warranted to determine how miRNAs block the assembly of the eIF4F translation initiation complex (Fukaya, 2014).

Functionally diverse microRNA effector complexes are regulated by extracellular signaling

Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. This study found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities (Wu, 2013).

This study found that different miRISC complexes are present in S2 cells, depending upon extracellular signals received by the cells. A constitutive G-miRISC complex composed of Ago1, miRNA, and GW182 is present under all signaling conditions tested. Other groups have shown that G-miRISC in S2 cells suppresses target mRNAs via inhibition of translation initiation and enhanced mRNA decay. This study found that lipid signaling does not affect G-miRISC but blocks other miRISC complexes from forming. This signaling is likely mediated by PKC because a phorbol ester mimics the effect of lipids on miRISC formation. Signaling blocks the formation of P-miRISC, which contains Ago1, miRNA, and Loqs-PB, but not GW182. P-miRISC represses translation of target mRNAs, which is manifested in polysome association of the complex. Thus, this work reveals a mechanistic shift in miRISC-executed translation repression under the influence of extracellular lipid signals. In the presence of lipid signaling, initiation is inhibited, and this occurs by G-miRISC. In the absence of lipid signaling, it is proposed that cells generate two levels of translational repression: one mediated by G-miRISC that inhibits initiation, and one mediated by P-miRISC that inhibits elongation. It is proposed that each miRISC complex independently represses the same target, and because they act in series (initiation - elongation), the net result on protein synthesis is the product (not sum) of each inhibitory step. This would provide the strongly synergized repression of reporter protein synthesis that was observed after serum withdrawal (Wu, 2013).

P-miRISC resembles the miRNA loading complex (miRLC) complex in terms of subunit composition (Ago1, Loqs-PB), but the two differ in one important way. Whereas miRLC contains premiRNA, P-miRISC contains mature miRNA. Thus, P-miRISC has an inherent potential to engage target mRNAs via base pairing interactions. It is suggested that P-miRISC is formed by the processing and loading of mature miRNA into Ago1 within the miRLC. Rather than releasing Loqs-PB/Dcr-1 and recruiting GW182, the loaded Ago1 retains Loqs-PB and never recruits GW182. P-miRISC can then engage target mRNAs, but its subunit composition dictates a different mode of repression upon the target (Wu, 2013).

Although GW182 and Loqs-PB binding to Ago1 are mutually exclusive, P-miRISC is not simply a default state when GW182 recruitment fails to occur. Knockdown of GW182 was insufficient to induce formation of P-miRISC. Moreover, formation of P-miRISC did not appear to occur at the expense of G-miRISC levels, as measured in sedimentation and immunoprecipitation experiments. This suggests a mechanism in which stable loading of miRNA is limited by the availability of cofactors for Ago1. Under serum-fed conditions, only GW182 is available, whereas both GW182 and Loqs-PB are available under serum-free conditions. This possibly offers a rapid way to modulate miRISC levels without the need for synthesis of more cofactors (Wu, 2013).

The switch in miRISC formation is regulated by PKC, but how this switch occurs is not clear. A recent study demonstrated that the mammalian homolog of Drosophila Ago1 can be phosphorylated by Akt3, which contributes to increased miRISC-mediated translation repression (Horman, 2013). However, no evidence was found for differential phosphorylation of Ago1 in S2 cells. A study of the mammalian ortholog of Loqs-PB, called TRBP, found it to be phosphorylated by ERK kinase in response to PKC. Phosphorylation stabilized miRLC and increased processing of growth-promoting miRNAs. The same mechanism was not shown for Loqs-PB, and examination of the Loqs-PB sequence failed to find strict conservation of those sites (Wu, 2013).

A second Ago1 complex also appears when lipid signaling is absent. Membrane-associated Ago1 likely contains miRNA, but not Loqs-PB or GW182. Association of mammalian Ago proteins with late endosomes has been previously observed. Drosophila Ago1 has also been observed to associate with endosomes in vivo. Endosomes have been proposed to serve as sites for miRISC turnover whereby miRISC continuously associates and releases from endosomes, constituting a mechanism that promotes miRISC recycling onto new targets. Thus, membrane-associated Ago1 may represent an intermediate in miRISC turnover. If so, where does the membrane- associated Ago1 originate? Several lines of evidence suggest that it originates from P-miRISC. First, its appearance precisely correlates with P-miRISC. Second, it is sensitive to puromycin treatment, which also disrupts association of P-miRISC with polysomes. However, membrane-associated Ago1 does not sediment in ribosome-containing fractions. Third, insulin specifically inhibits membrane-associated Ago1, arguing that membrane-associated Ago1 is not an obligate precursor of P-miRISC. The simplest interpretation of these data is that membrane- associated Ago1 is formed from a P-miRISC precursor. If so, then Loqs-PB dissociation must be involved in the conversion because Loqs-PB is not found in the membrane-associated complex. A similar manner of cofactor stripping was observed for GW182, which dissociated from Ago-miRNA complexes when they associated with endosomes. Perhaps, cofactor dissociation is a fundamental part of the recycling mechanism (Wu, 2013).

This model might provide some insights into a long-standing controversy in the miRNA field. Some studies have found evidence for translation initiation as the regulated step, whereas others have found evidence for translation elongation. This work provides a potential explanation for these differences. That is, experimental model systems experiencing diverse extracellular signals might respond accordingly to form distinct types of miRISC complexes, which regulate different steps of translation. Thus, all studies have depicted an accurate picture of miRISC activity because signals that dictate miRISC subunit composition affect its mode of action (Wu, 2013).

Mei-P26 regulates the maintenance of ovarian germline stem cells by promoting BMP signaling

In the Drosophila ovary, bone morphogenetic protein (BMP) ligands maintain germline stem cells (GSCs) in an undifferentiated state. The activation of the BMP pathway within GSCs results in the transcriptional repression of the differentiation factor bag of marbles (bam). The Nanos-Pumilio translational repressor complex and the miRNA pathway also help to promote GSC self-renewal. How the activities of different transcriptional and translational regulators are coordinated to keep the GSC in an undifferentiated state remains uncertain. Data presented in this study show that Mei-P26 cell-autonomously regulates GSC maintenance in addition to its previously described role of promoting germline cyst development. Within undifferentiated germ cells, Mei-P26 associates with miRNA pathway components and represses the translation of a shared target mRNA, suggesting that Mei-P26 can enhance miRNA-mediated silencing in specific contexts. In addition, disruption of mei-P26 compromises BMP signaling, resulting in the inappropriate expression of bam in germ cells immediately adjacent to the cap cell niche. Loss of mei-P26 results in premature translation of the BMP antagonist Brat in germline stem cells. These data suggest that Mei-P26 has distinct functions in the ovary and participates in regulating the fates of both GSCs and their differentiating daughters (Li, 2012).

Evidence is provided that Mei-P26 promotes GSC self-renewal in addition to its previously described role in negatively regulating the miRNA pathway during germline cyst development. Disruption of mei-P26 results in a bam-dependent GSC loss phenotype and further characterization reveals that Mei-P26 fosters BMP signal transduction within GSCs by repressing Brat protein expression. In addition, Mei-P26 also appears to participate in the miRNA-mediated silencing of orb mRNA in GSCs. These results indicate that Mei-P26 carries out multiple functions within the Drosophila ovary and might be at the center of a molecular hierarchy that controls the fates of GSCs and their differentiating daughters (Li, 2012).

Three observations suggest that mei-P26 functions within GSCs. First, the average number of GSCs per terminal filament decreases from an average of two to well below one in mei-P26 mutant ovaries. Second, mei-P26 mutant germline clones are rapidly lost from the GSC niche. Third, syncytial cysts and Bam-expressing cells are often observed immediately adjacent to the cap cells in mei-P26 mutant ovaries (Li, 2012).

Research over the last ten years has shown that BMP ligands emanating from cap cells at the anterior of the germarium initiate a signal transduction cascade in GSCs that results in the transcriptional repression of bam. Stem cell daughters one cell diameter away from the cap cell niche express bam, suggesting that a steep gradient of Dpp availability or responsiveness exists between GSCs and cystoblasts. Recent work has shed light on how various mechanisms antagonize BMP signaling in cystoblasts. For example, the ubiquitin ligase Smurf (Lack -- FlyBase) promotes germline differentiation and partners with the serine/threonine kinase Fused to reduce levels of the Dpp receptor Tkv in cystoblasts. The TRIM-NHL domain protein Brat also functions in cystoblasts, serving to translationally repress Mad expression. Notably, inappropriate expression of Brat within GSCs results in a stem cell loss phenotype. Brat itself is translationally repressed in GSCs by the Pumilio-Nanos complex. Mutant phenotypes and co-IP experiments presented in this study support a model in which Mei-P26 partners with Nanos to repress Brat expression in GSCs. This negative regulation of Brat expression protects the BMP signal transduction pathway in GSCs from inappropriate deactivation (Li, 2012).

Mei-P26 appears to enhance miRNA-dependent translational silencing within GSCs based on several lines of experimental evidence. First, co-IP experiments using ovarian extracts from c587-gal4>UAS-dpp and bam mutants suggest that Mei-P26 physically associates with Ago1 and GW182 in undifferentiated germ cells. Second, disruption of mei-P26 results in a GSC loss phenotype, similar to the effects of disrupting components of the miRNA pathway tested to date. Third, Mei-P26 and Ago1 can physically associate with the same target mRNA. Finally, disruption of either Ago1 or mei-P26 results in increased expression of this target in GSCs. The evidence that Mei-P26 promotes miRNA action in certain contexts is consistent with the established activities of its close homologs NHL-2 and TRIM32 (Li, 2012 and references therein).

It is proposed that Mei-P26 regulates GSC self-renewal and early germ cell differentiation through distinct mechanisms. In GSCs, Mei-P26 promotes self-renewal by repressing the expression of Brat and potentially other negative regulators of BMP signal transduction. Within stem cells, Mei-P26 also functions together with miRISC to attenuate the translation of specific mRNAs. miRISC does not appear to target brat mRNA based on clonal data. However, the possiblity cannot be ruled out that the enhancement of miRNA-mediated silencing of some mRNAs by Mei-P26 contributes to stem cell self-renewal. Interestingly, recent findings suggest that Pumilio can function together with the miRNA pathway in certain contexts In BJ primary fibroblasts, Pumilio 1, miR-221 and miR-222 regulate the expression of p27 in a 3' UTR-dependent manner. In response to growth factors, Pumilio 1 becomes phosphorylated, which in turn increases its RNA binding activity. Pumilio 1 binding to p27 mRNA results in a conformational change in the 3' UTR that allows miR-221 and miR-222 to bind more efficiently, resulting in greater repression of p27. Perhaps, together, Drosophila Pumilio, Nanos, Ago1 and Mei-P26 also silence specific messages in specific contexts. Identifying more direct in vivo targets for these proteins within GSCs will be crucial for testing this idea (Li, 2012).

In cystoblasts, Mei-P26 promotes germline cyst development by antagonizing the miRNA pathway. This study shows that Mei-P26 can also promote miRNA translational repression in another cell, the GSC. Evidence is provided that Mei-P26 physically associates with miRISC and co-regulates translation of at least one mRNA, orb, through specific elements within its 3′UTR. In cystoblasts and early developing cysts, the induction of Bam expression might cause Mei-P26 to switch from an miRISC-associated silencer to an miRNA antagonist. How Bam activates this switch is currently under investigation. The finding that Mei-P26 functions in both GSCs and differentiating cysts hints at a mechanism whereby different translational repression programs coordinate changes in cell fate (Li, 2012).

Further work will be needed to determine the specific biochemical function of Mei-P26 when it associates with either the Nanos complex or miRISC. Like other TRIM-NHL domain proteins, Mei-P26 contains a RING domain that may have E3 ubiquitin ligase activity. Based on results presented in this study, it is proposed that Mei-P26 and perhaps other TRIM-NHL domain proteins act as effectors for multiple translational repressor complexes. In this model, Mei-P26 is targeted to specific mRNAs through sequence-directed RNA-binding proteins. Specific protein substrates of Mei-P26 in the germline have not yet been determined but identifying these targets will provide key insights into how Mei-P26 and other related TRIM-NHL domain proteins regulate translational repression. Furthermore, the Mei-P26 complex is likely to target additional mRNAs for silencing in both GSCs and developing cysts. Identifying more of these mRNAs will further elucidate the complex translational regulatory hierarchies that control the balance between stem cell self-renewal and differentiation (Li, 2012).

The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, this study characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

Recent studies indicate that translational repression of miRNA targets precedes deadenylation and decay. This study shows that these two functional outcomes of miRNA regulation are linked and both require the interaction of GW182 proteins with PABP and deadenylases (Huntzinger, 2013).

The interaction of GW182 proteins with PABP has been well documented using biochemical and structural studies, and the PAM2 motif is highly conserved among vertebrate and insect GW182 proteins. Despite conservation, the study of the role of PABP in silencing in different systems has led to conflicting conclusions. For example, several studies have reported that the PABP–GW182 interaction is important for silencing in Drosophila and human cells and in cell-free systems that recapitulate silencing. Furthermore, PABP depletion prevented miRNA-mediated deadenylation in cell-free extracts from mouse Krebs-2 ascites cells, and mutations in the PAM2 motif of TNRC6C reduced the rate of deadenylation in tethering assays. In addition, a study in Drosophila cell-free extracts wherein silencing is mediated through endogenous preloaded miRISCs indicated that PABP stimulates silencing by facilitating the association of miRISC complexes with mRNA targets. It was also shown that on miRISC binding, PABP progressively dissociated from the mRNA target, in the absence of deadenylation (Huntzinger, 2013).

In contrast to the studies mentioned above, studies in zebrafish embryos and in a Drosophila cell-free assay wherein miRISCs are loaded with exogenously supplemented miRNA duplexes indicate that PABP is dispensable for miRNA-mediated silencing. Intriguingly, efficient silencing in zebrafish embryos required the GW182 PAM2 motif. Moreover, the observation that multiple and non-overlapping fragments of Drosophila GW182 (including N-term fragments that do not interact with PABP) silenced mRNA reporters in tethering assays was interpreted as evidence that the interaction of GW182 proteins with PABP is not required for silencing. This study shows that unlike in tethering assays, N-term fragments of GW182 fail to restore the silencing of a majority of the reporters tested in complementation assays. Thus, tethering assays bypass the requirement for PABP binding, and may not faithfully recapitulate silencing. Furthermore, the observation that PABP dissociates from the poly(A) tail of miRNA targets in the absence of deadenylationprovides one explanation for the occurrence of silencing in extracts in which PABP has been depleted or displaced from the poly(A) tail using an excess of Paip2 (Huntzinger, 2013).

In summary, these results confirm and further extend previous observations that a single amino acid substitution in the PAM2 motif of human TNRC6 proteins abolishes PABP binding and impairs silencing activity, despite the interaction of this mutant with deadenylases. Furthermore, Drosophila GW182 N-term protein fragments that bind deadenylases, but not PABP, failed to complement the silencing of eight of the nine reporters tested, although they are active in tethering assays. These results provide evidence for a role of PABP in silencing in human and Drosophila cells. However, it is possible that PABP becomes dispensable for silencing depending on cellular conditions or the nature of the specific mRNA target, as shown, for example, for the F-Luc-Nerfin-1 reporter when silencing is mediated by miR-9b (Huntzinger, 2013).

The SDs of human TNRC6 proteins directly interact with CNOT1 through tryptophan-containing motifs in the M1, M2 and C-term regions of the S. This study shows that these motifs contribute additively to CNOT1 binding and silencing activity in human cells. Indeed, when at least two motifs are simultaneously mutated, CNOT1 binding is strongly reduced and silencing activity impaired (Huntzinger, 2013).

The interaction between GW182 and deadenylases is conserved in Drosophila; however, in contrast to human SDs, the Drosophila SD is not sufficient for NOT1 binding. This study shows that in addition to the SD, the Q-rich region is required for full NOT1 binding activity. Thus, although Drosophila GW182 has lost the CIM-2 motif, this protein has acquired additional motifs that can interact with NOT1. This study also shows that in contrast to the human proteins, Drosophila GW182 can interact with NOT2 and PAN3 via N-term sequences. Consequently, Drosophila GW182 can recruit deadenylases in multiple ways. Considering that (1) NOT1 interacts with NOT2, (2) the PAN2–PAN3 complex interacts with PABP and (3) the CCR4–NOT and PAN2–PAN3 complexes form a larger multiprotein complex in vivo, the current observations indicate a high degree of connectivity and redundancy within the GW182 interaction network, which could explain why mutations in individual motifs do not abolish partner binding or silencing activity, but a combination of two or more mutations is required to abrogate binding and silencing activity (Huntzinger, 2013).

In addition, the ability of Drosophila GW182 N-term fragments to bind deadenylases also explains why these fragments are potent triggers of translational repression and mRNA degradation in tethering assays, whereas the corresponding fragments of the human proteins exhibit only residual activity. As discussed previously, despite their activity in tethering assays, Drosophila GW182 N-term fragments failed to complement the silencing of several of the reporters tested. The reason for the different activities of these fragments in tethering and complementation assays remains unknown (Huntzinger, 2013).

This study has demonstrated that silencing (i.e. translational repression and target degradation) requires the interaction between GW182 proteins and both PABP and deadenylases. Several lines of evidence support this conclusion. First, the TNRC6C SD, which is sufficient for PABP and deadenylase binding, rescues silencing when fused to a minimal ABD. Similarly, the minimal fragment of Drosophila GW182 that rescues silencing comprises the Q+SD region, which also binds both deadenylases and PABP. Second, the Drosophila GW182 N-term fragments that bind deadenylases but not PABP are generally inactive in complementation assays. Third, mutations that specifically disrupt TNRC6 binding to PABP or deadenylase impair silencing, and mutations that disrupt deadenylase binding exhibit a stronger deleterious effect. Silencing activity is abolished when these mutations are combined. Finally, silencing is inhibited in human cells overexpressing the CNOT1 Mid domain together with a catalytically inactive CNOT7 mutant. In combination with the previously published data, these results indicate that silencing minimally requires an AGO, a GW182 protein, PABP and deadenylases, thus defining the minimal interaction network required for silencing. The findings do not rule out that additional interactions are potentially required to achieve maximal repression, depending on the cellular context or the mRNA target. For example, the P-GL motif is highly conserved and important for silencing in zebrafish embryos. This motif may mediate interactions with additional partners (Huntzinger, 2013).

The finding that deadenylase complexes, in particular, are required for miRNA-mediated translational repression has broad implications regarding post-transcriptional mRNA regulation. Indeed, in addition to the GW182 proteins, various sequence-specific mRNA-binding proteins, such as Nanos, Bicaudal-C and Pumilio, recruit the CCR4–NOT complex to their mRNA targets. Furthermore, the direct tethering of the subunits of the CCR4–NOT complex represses the translation of mRNA reporters lacking a poly(A) tail, suggesting that the CCR4–NOT complex promotes translational repression in the absence of deadenylation. Therefore, elucidating the mechanism by which the CCR4–NOT complex regulates the fates of mRNA targets promises to increase understanding of the mechanism underlying repression by miRNAs and diverse sequence-specific RNA-binding proteins (Huntzinger, 2013).

A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. This study elucidated the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. The conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. It was further shown that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, these data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping (Chen, 2014).

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing; Dicer-2 is required for P-body integrity

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. This study shows that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity (Eulalio, 2007).

The first proteins found in P bodies are those functioning in the degradation of bulk mRNA. In eukaryotes, this process is initiated by removal of the poly(A) tail by deadenylases. There are several deadenylase complexes in eukaryotes: the PARN2-PARN3 complex is thought to initiate deadenylation, which is then continued by the CAF1-CCR4-NOT complex. Following deadenylation, mRNAs are exonucleolytically digested from their 3' end by the exosome, a multimeric complex with 3'-to-5' exonuclease activity. Alternatively, the cap structure is removed by the decapping enzyme DCP2 after deadenylation, rendering the mRNA susceptible to 5'-to-3' degradation by the major cytoplasmic exonuclease XRN1 (Eulalio, 2007).

Decapping requires the activity of several proteins generically termed decapping coactivators, though they may stimulate decapping by different mechanisms. In the yeast Saccharomyces cerevisiae, these include DCP1, which forms a complex with DCP2 and is required for decapping in vivo, the enhancer of decapping-3 (EDC3 or LSm16), the heptameric LSm1-7 complex, the DExH/D-box RNA helicase 1 (Dhh1, also known as RCK/p54 in mammals), and Pat1, a protein of unknown function that interacts with the LSm1-7 complex, Dhh1, and XRN1. In human cells, DCP1 and DCP2 are part of a multimeric protein complex that includes RCK/p54, EDC3, and Ge-1 (also known as RCD-8 or Hedls), a protein that is absent in S. cerevisiae (Eulalio, 2007).

The decapping enzymes, decapping coactivators, and XRN1 colocalize in P bodies. Additional P-body components in multicellular organisms include the protein RAP55 (also known as LSm14; Drosophila homolog - Trailer hitch), which has a putative role in translation regulation, and GW182, which plays a role in the microRNA (miRNA) pathway (Eulalio, 2007).

The P-body marker GW182 localizes to cytoplasmic foci in Drosophila S2 cells together with the decapping enzyme DCP2 and the decapping coactivator DCP1, suggesting that these foci represent P bodies. To characterize D. melanogaster P bodies further, antibodies were raised to the Drosophla orthologs of two proteins found in human-cell P bodies. These correspond to Ge-1 and Tral (LSm15), which is closely related to human RAP55 (or LSm14) (see Tanaka, 2006). Both antibodies stained the cytoplasm diffusely and also stained discrete cytoplasmic foci with a diameter ranging from 100 nm to 300 nm. The antibody signals are specific, as they are lost in cells in which the cognate proteins were depleted. The foci are present in about 95% of the cell population and are readily detectable because the concentration of Tral or Ge-1 in these foci is significantly higher than that in the surrounding cytoplasm (Eulalio, 2007).

The distribution of green fluorescent protein (GFP)-tagged versions of proteins found in P bodies was examined in yeast and/or human cells. These include DCP1, DCP2, GW182, Me31B (the D. melanogaster ortholog of S. cerevisiae Dhh1 and vertebrate RCK/p54), CG5208 (the D. melanogaster homolog of S. cerevisiae Pat1, referred to as HPat hereafter), and EDC3 (also known as LSm16). All of these proteins formed cytoplasmic foci that costained with the anti-Tral or anti-Ge-1 antibodies. Importantly, the expression of the GFP-tagged proteins did not significantly alter the number and size of endogenous P bodies. Together, these results indicate that the localization of decapping enzymes and decapping coactivators into P bodies is evolutionarily conserved. The localization of GW182 in Drosophila P bodies is in agreement with the proposal that GW-bodies and P bodies overlap, as reported for mammalian cells (Eulalio, 2007).

The localization of proteins implicated in translational regulation was examined in Drosophila oocytes whose corresponding transcripts are detectable in S2 cells, in particular, Smaug and the dsRNA binding protein Staufen. Smaug is a translational repressor that also promotes deadenylation of bound mRNAs by recruiting the CAF1-CCR4-NOT1 complex (Zaessinger, 2006). Both proteins localized to P bodies with endogenous Tral. Strikingly, P bodies increased in size in cells expressing Staufen at high levels but not in cells overexpressing GFP fusions of Smaug, suggesting that Staufen promotes P-body formation. Drosophila Staufen, Tral, DCP1, DCP2, XRN1, and Me31B have also been detected in RNP granules in neuronal cells and/or in oocytes, indicating that P bodies and other RNP granules observed in neuronal cells or during development share common components (Eulalio, 2007).

P-body formation requires nontranslating mRNPs and/or mRNPs undergoing decapping. A conserved feature of P bodies in human and yeast cells is that their formation depends on RNA and is enhanced in cells in which the concentration of nontranslating mRNAs or of mRNAs undergoing decapping increases. These observations indicate that mRNAs must exit the translation cycle to localize to P bodies. In agreement with this, it was observed that Drosophila P bodies decline when cells are treated with RNase A or with cycloheximide (which inhibits translation elongation and stabilizes mRNAs into polysomes). In contrast, P-body sizes increase in cells treated with puromycin, which causes premature polypeptide chain termination and polysome disassembly. Both puromycin and cycloheximide inhibit protein synthesis in S2 cells, as judged by the reduction of F-Luc and R-Luc activities after the treatment of cells transiently expressing these proteins with these drugs (Eulalio, 2007).

The size of Drosophila P bodies also depends on the fraction of mRNAs undergoing decapping, in agreement with the results reported for yeast and human cells. Indeed, blocking mRNA decay at an early stage, for instance, by preventing deadenylation in cells in which NOT1 (a component of the CAF1-CCR4-NOT deadenylase complex) is depleted, leads to the dispersion of P bodies, whereas P bodies are on average more prominent in cells from which DCP2 or XRN1 is depleted (in which decapping and subsequent 5'-to-3' mRNA decay are inhibited) (Eulalio, 2007).

Several lines of evidence show that P bodies do not serve as storage sites for the effectors of posttranscriptional process but are sites where mRNA degradation and silencing can take place. For instance, P-body formation is RNA dependent, and decay intermediates, siRNAs, and miRNAs and their targets are detected in P bodies. Moreover, the size and number of P bodies depends on the fraction of mRNAs undergoing decapping. However, the question of whether mRNA decay and silencing require the environment of microscopic, wild-type P bodies to occur or whether these processes can also occur outside of P bodies in soluble protein complexes remains open. This study shows that formation of large P bodies visible in the light microscope as observed in wild-type cells is not required for several processes associated with P-body components, including NMD, mRNA decay, and RNA-mediated gene silencing (Eulalio, 2007).

The question addressed in this study was whether the environment of macroscopic P bodies is required for posttranscriptional regulation. P bodies are defined as the large cytoplasmic foci visible by light microscopy in wild-type cells. These foci are on average 100 to 300 nm in diameter and are readily detected as bright cytoplasmic dots because the concentration of proteins in these foci is significantly higher than in the surrounding cytoplasm. Nevertheless, most P-body components are also detected diffusely throughout the cytoplasm. For a limited number of examples that have been analyzed, it has been shown that P-body components are not confined to these structures but dynamically exchange with the cytoplasmic pool. Quantitative information regarding the fractionation of P-body components between P bodies and the cytoplasm is still lacking, but given the volume of P bodies relative to that of the cytoplasm, it is likely that the diffuse cytoplasmic fraction is significantly larger. This suggests that posttranscriptional processes are likely to occur and may even be initiated in the diffuse cytoplasm or in soluble protein complexes that aggregate to form P bodies. Whether these processes take place in submicroscopic aggregates or soluble protein complexes in the absence of detectable microscopic P bodies remains to be solved. However, it is considered that aggregates or large multiprotein assemblies that are not detectable by light microscopy cannot be defined as bodies (Eulalio, 2007).

Translation factors or ribosomes are generally not present in P bodies (with the exception of cap binding protein eIF4E), indicating that mRNAs leave the translation cycle prior to entering P bodies. Consistently, releasing mRNAs from polysomes leads to increases in P-body sizes and numbers, whereas the stabilization of mRNAs into polysomes disrupts P bodies. These observations suggest that a critical step in P-body formation is the release of mRNPs from a translationally active state associated with polysomes to a translationally inactive state. This paper has shown that releasing mRNAs from polysomes by puromycin treatment is not sufficient to elicit P-body formation and that functional silencing pathways or proteins generically termed decapping coactivators are required for P-body assembly. These proteins include Me31B (Dhh1 in yeast), HPat (Pat1 in yeast), Ge-1, and the LSm1-7 complex (Eulalio, 2007).

What could be the role of these proteins in P-body formation? Me31B is an RNA helicase which could facilitate rearrangements in mRNP composition upon release from polysomes. The role of HPat is unclear, but the yeast ortholog interacts with Dhh1, XRN1, and the heptameric LSm1-7 complex. Coimmunoprecipitation assays indicate that the interaction between Dhh1 and Pat1 orthologs (i.e., Me31B and HPat) is conserved in Drosophila. Finally, the LSm1-7 complex associates with deadenylated mRNAs and stimulates decapping. Clearly, many details regarding the precise molecular function of these proteins remain to be discovered, but their requirement for P-body assembly indicates that mRNAs that are not actively translated do not enter into P bodies by default: the activity of a defined set of proteins is required. Alternatively, nontranslating mRNAs may enter silencing pathways, and this would also lead to changes in mRNP composition due to the recruitment of Argonaute proteins and binding partners, which include P-body components such as GW182, decapping enzymes, and RCK/p54 (Eulalio, 2007).

Once P-body components are bound to an RNP, P-body formation may then be triggered by protein-protein interactions. Indeed, proteins required for P-body assembly are known to interact to form multimeric protein complexes. Consistently, in addition to the interactions mentioned above, DCP1, DCP2, Ge-1, RCK/p54, and EDC3 form a multimeric protein complex in human cells. The absolute requirement of RNA for P-body formation could be explained if affinities between these proteins increased upon RNA binding. Additionally, proteins like GW182 and Ge-1 are multidomain proteins that could bind more than one RNP simultaneously, bringing into close proximity several components and thus nucleating the formation of P bodies (Eulalio, 2007).

RNAs targeted by silencing pathways nucleate P bodies. In this study, it is shown that both the RNAi and miRNA pathways contribute to the generation of a pool of nontranslating mRNPs and/or of mRNPs committed to decay which are required for P-body formation. Nevertheless, silencing can occur in the absence of microscopic P bodies. The results provide support to previous models proposing that silencing is initiated in the cytoplasm and that the localization of the silencing machinery into P bodies is a consequence, rather than the cause, of silencing (Eulalio, 2007).

An unexpected observation from these studies is that AGO2 and Dicer-2, which function in siRNA-mediated gene silencing in Drosophila, are required for P-body integrity. The role of these proteins in P-body assembly is unlikely to be structural, because P bodies are restored upon puromycin treatment in cells from which AGO2 or Dicer-2 is depleted. The most likely explanation for the requirement of these proteins is, therefore, that silencing by siRNAs also generates RNPs that elicit P-body formation. The requirement for AGO2 could be at least partially explained by the observation that the expression levels of a small subset of endogenous miRNA targets are affected in AGO2-depleted cells, suggesting that some miRNAs may be loaded into AGO2-containing RNA-induced silencing complexes. Furthermore, the AGO1 and AGO2 genes interact, although it is unclear how this interaction affects the activities of these proteins (Eulalio, 2007).

The requirement for Dicer-2 in P-body assembly, however, suggests that endogenous siRNA targets also contribute to P-body formation. Because the levels of dsRNA synthesis from endogenous loci that could provide precursors for the production of endogenous siRNAs are currently unknown, the fraction and origin of transcripts regulated by endogenous siRNAs cannot be estimated. Nonetheless, a possible source of endogenous dsRNAs is the bidirectional transcription of pseudogenes and transposable elements, in agreement with the role of the RNAi pathway as a defense mechanism against RNA viruses and mobile genetic elements (Eulalio, 2007).

The essential role of silencing pathways in P-body formation in Drosophila, and presumably in human cells, raises the question of how P bodies are assembled in S. cerevisiae, which lacks silencing pathways. One possibility is that other posttranscriptional processes generate nontranslating mRNPs required to nucleate P bodies. For instance, the NMD pathway contributes to P-body assembly in yeast cells, because depletion of Upf2 or Upf3 leads to increases in P-body size and number in a Upf1-dependent manner, whereas similar experiments with Drosophila cells do not affect P bodies (Eulalio, 2007).

With the exception of the proteins involved in silencing, the composition of P bodies and the effects of drugs such as cycloheximide and puromycin on P-body size and number are strikingly similar in yeast, Drosophila, and human cells, raising the question of what the role of these structures accounting for their conservation in eukaryotic cells could be. The results show that the environment of microscopic P bodies is not essential for mRNA decay or silencing but do not exclude that the formation of P bodies confers a kinetic advantage. Moreover, the results do not rule out a role for large P bodies in sequestering a specific set of nontranslating mRNPs and reinforcing their repression by shielding them from the translation machinery (Eulalio, 2007).

Finally, the conservation of P bodies may reflect a role for these structures in other cellular processes that is not yet fully appreciated. A role in some steps of retroviral or retrotransposon life cycles is suggested by the localizations of the antiretroviral proteins APOBEC3G and APOBEC3F in human cell P bodies and of the protein and RNA components of the retrovirus-like element Ty3 in yeast P bodies. A link between P bodies and the regulation of retrotransposition would be consistent with the role of RNAi pathways in silencing the expression of transposable elements. Because all known essential P-body components play roles in decapping and/or silencing and proteins playing an exclusively structural role in P-body assembly have not yet been identified, it is currently not possible to evaluate the role of P bodies for cell, tissue, or organism survival (Eulalio, 2007).

Search PubMed for articles about Drosophila GW182/Gawky

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date revised: 7 October 2021

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